Number of New Cases and Deaths Per 100,000 People (All Races, Females), Age -Adjusted

**Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1975 105.1 - 31.4 1976 101.9 - 31.8 1977 100.8 - 32.5 1978 100.6 - 31.7 1979 102.1 - 31.2 1980 102.3 - 31.7 1981 106.4 - 31.9 1982 106.5 - 32.2 1983 111.1 - 32.1 1984 116.0 - 32.9 1985 124.3 - 33.0 1986 126.9 - 32.9 1987 134.5 - 32.7 1988 131.3 - 33.2 1989 127.2 - 33.2Number of New Cases and Deaths Per 100,000 People (All Races, Females), Age -Adjusted

**Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1990 131.9 - 33.1 1991 133.8 - 32.7 1992 132.1 130.0 31.6 1993 129.2 127.2 31.4 1994 131.0 128.8 30.9 1995 132.6 130.9 30.6 1996 133.7 132.1 29.5 1997 138.0 136.0 28.2 1998 141.4 139.0 27.5 1999 141.5 138.5 26.6 2000 136.5 134.2 26.6 2001 138.8 135.8 26.0 2002 135.7 132.7 25.6 2003 126.9 124.0 25.3 2004 128.0 124.8 24.5Number of New Cases and Deaths Per 100,000 People (All Races, Females), Age -Adjusted

**Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  2005 126.5 124.3 24.1 2006 126.1 122.7 23.6 2007 128.1 125.9 23.0 2008 128.1 126.0 22.6 2009 130.5 127.4 22.2 2010 126.5 122.9 21.9 2011 129.6 126.2 -**Figure 1.5: Estimated Breast Cancer Incidence (New Cases) Rates among Women**  **by**

**State, 2006 -** **2010 State**  **Rate of Invasive**

**Breast Cancer (per 100,000**  **women) State**  **Rate of Invasive Breast Cancer**  **(per 100,000 women)**  United States 122 Missouri 122 Alabama 119 Montana 124 Alaska 128 Nebraska 122 Arizona 110 Nevada 113 Arkansas 110 New Hampshire 132 California 122 New Jersey 129 Colorado 125 New Mexico 109 Connecticut 136 New York 128 Delaware 127 North Carolina 125 District of Columbia 140 North Dakota 123 Florida 114 Ohio 121 Georgia 122 Oklahoma 122 Hawaii 123 Oregon 130Idaho 120 Pennsylvania 126

Illinois 126 Rhode Island 131 Indiana 117 South Carolina 122 Iowa 123 South Dakota 118 Kansas 123 Tennessee 129 Kentucky 121 Texas 114 Louisiana 120 Utah 111 Maine 127 Vermont 131 Maryland 128 Virginia 125 Massachusetts 134 Washington 131 Michigan 120 West Virginia 110 Minnesota Not available Wisconsin 123 Mississippi 114 Wyoming 111 Source: American Cancer Society, 2014 [ 37 ]EPIDEMIOLOGY Exposure to polychlorinated biphenyl (PCB) congeners measured shortly after giving birth and subsequent risk of maternal breast cancer before age 50 Barbara A. Cohn • Mary Beth Terry • Marj Plumb • Piera M. Cirillo Received: 24 July 2012/Accepted: 12 September 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Discrete windows of susceptibilit y to toxicants have been identi fied for the breast, including in utero, puberty, pregnancy, and postpartum. We tested the hypothesis that polychlorinated biphenyls (PCBs) mea sured during the early postpartum predict increased risk of maternal breast cance r diagnosed before age 50. We ana - lyzed archived early postpartum serum samples collected from 1959 to 1967, an average of 17 years before diagnosis (mean diagnosis age 43 years) for 16 PCB congeners in a nested case–control study in the Child Health and Devel - opment Studies cohort (N = 112 cases matched to controls on birth year). We used conditional logistic regression to adjust for lipids, race, year, lactation, and body mass. We observed strong breast cancer associations with three congeners. PCB 167 was associated with a lower risk (odds ratio (OR), 75th vs. 25th percentile = 0.2, 95 % con fi - dence interval (95 % CI) 0.1, 0.8) as was PCB 187 (OR, 75th vs. 25th percentile = 0.4, 95 % CI 0.1, 1.1). In con - trast, PCB 203 was associated with a sixfold inc reased risk (OR, 75th vs. 25th percentile = 6.3, 95 % CI 1.9, 21.7). The net association of PCB exposure, estimated by a post - hoc score, was nearly a threefold increase in risk (OR, 75th vs. 25th percentile = 2.8, 95 % CI 1.1, 7.1) among women with a high er proportion of PCB 203 in relation to the sum of PCBs 167 and 187. Postpartum PCB exposure likely also represents pregnancy exposure, and may predict increased risk for early breast cancer depending on the mixture that represents internal dose. It remain s unclear whether individual differences in exposure, response to exposure, or both explain risk patterns observed. Keywords PCBs Polychlorinated biphenyls Breast cancer Pregnancy Postpartum Prospective Human Abbreviations OR Odds ratio PCB(s) Polychl orinated biphenyl(s) 95 % CI 95 percent con fidence interval Introduction Discrete windows of susceptibility to toxicants have been identi fied for the breast, including in utero, during puberty, and during pregnancy and postpartum [1–4]. Human studies of brea st cancer have not been able to assess the effects of measured exposure to environmental chemicals during windows of susceptibility for the breast [2, 5]. The existing literature on polychlorinated biphenyls (PCBs) and breast cancer typically has focused p rimarily on postmen - opausal breast cancer [6–8]. In addition, exposure in prior studies was largely measured in middle age or at time of diagnosis, was variable on how PCBs were classi fied and B. A. Cohn (&) P. M. Cirillo Child Health and Development Stud ies, Public Health Institute, 1683 Shattuck Avenue,Ste. B, Berkeley, CA 94709, USA e - mail: bcohn@chdstudies.org M. B. Terry Department of Epidemiology, Mailman School of Public Health of Columbia University, New York, NY, USA M. B. Terry Herbert Irving Co mprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA M. Plumb Plumbline Coaching and Consulting, Inc., 2759 Park Street, Berkeley, CA 94702, USA 123 Breast Cancer Res Treat DOI 10.1007/s10549 - 012 - 2257 - 4 whether individual congen ers were considered, and was based on samples obtained after PCB use was regulated [6– 8].TheliteratureonthepesticideDDTandbreastcanceris subject to similar limitations [9]. Findings on the relation of PCBs measured in middle age to breast cancer are compl ex and have been reviewed previously in detail (see Table 3, pp. 2872 – 2876 in Brody et al. [8]). Brie fly, some studies reported positive associa - tions with individual congeners, others reported no asso - ciations for total PCBs or any individual congeners, and others reported negative associations with some tumor types. The most consistent, positive findings were reported where interaction between exposure and CYP1A1 gene polymorphisms could be considered. Three studies found higher PCB exposures with postmen opausal breast cancer in combination with the CYP1A1 polymorphism, M2 type [10– 12]andanotherreportedthisassociationwithpre - menopausal breast cancer [13]. The present prospective study adds a unique perspective by testing the hypothesis that exposure to PC Bs, measured during the early postpartum, is associated with increased risk of early breast cancer. Because of the long half- life of PCBs [14], early postpartum levels also re flect exposure during pregnancy [15] and possibly during childhood and adolescence . To our knowledge, this is the first study to report on breast cancer associations in relation to measured PCBs levels during critical periods of vulnerability for the breast. Materials and methods Subjects Subjects were participants in the Child Health an d Devel - opment Studies (CHDS), residents of the Oakland, Cali fornia area and members of the Kaiser Permanente Health Plan who sought obstetric care between 1959 and 1967 [16].SubjectsvoluntarilyparticipatedintheCHDS,giving an oral informed consent for a n in - person interview, col - lection of blood specimens at several points in pregnancy and the early postpartum, and permission for medical record access. This study was reviewed and approved by The Institutional Review Board of the Public Health Institute and we have complied with all federal guidelines governing use of human participants. Breast cancer cases were identi fied by linkage to the California Cancer Registry, and the California Vital Status Records [17]. All names for each CHDS subject are sub - mi tted for cancer linkages using fixed (i.e. birth date, sex, race, and name) and changeable (i.e. address and patient record number) identi fiers. A rigorous protocol is used toverify cases, comparing fixed versus changeable identifiers by manual review. The Ca lifornia Cancer Registry is reported to be [99 % complete after a lag time of about 2 years [18]. Cases were de fined as women with incident invasive or non - invasive breast cancer diagnosed before age 50, or deaths due to breast cancer before age 50, obtaine d from linkage conducted in early 1998. There were 133 cases who met study criteria. All members of the CHDS cohort are additionally linked to the California Department of Motor Vehicles (DMV) files on a regular basis to determine res - idence history allowi ng us to assess their control status and to update any name changes. All names registered with the DMV are used in establishing a match. Simultaneous linkage of multiple family members enhances matching. The regular DMV matching provides a history of locat ion for each subject which is used to determine the population at risk for cancer, corresponding with geographic surveil - lance by California’s cancer registries. Subjects who can - not be located are considered lost to follow - up at the date of their last d e finitive classification as a California resident. One control, matched exactly on birth year, was selected at random for each case from those who were under cancer surveillance and known to be free of breast cancer at the age of diagnosis for the matching c ase. The median time to diagnosis for cases was 17 years. The mean age at diag - nosis was 43 years. Serum assays Exposure to PCBs was measured by assays of serum samples drawn during the early postpartum period, within 1–3 days of delivery. Postpartum samp les were used to conserve valuable archived serum samples drawn in each trimester for future studies where timed samples are essential. PCBs have a long half- life and prior work has established stability of organochlorine levels assayed across all trimeste rs of pregnancy and the early postpartum [15].Forthisreason,postpartumlevelsofPCBsmay accurately rank women on their pregnancy exposure, covering two potential vulnerable periods of susceptibility of the breast to toxicants. PCBs were assayed in the labora tory of Dr. Mary Wolff [19] using modi fications of methods developed previously by Brock et al. [20]. Briefly, a polar extraction of serum lipids is followed by a column chromatographic clean - up and enrichment step, with analysis by gas chromatography with e lectron capture detection. Limits of detection were approximately 0.07 ng/mL for individual compounds based on three times the standard deviation of the levels found in the lowest quality control plasma pool [21]. When the serum pool and blanks were consid ered together [22], the limit of detection was 0.01 –0.1 ng/mL; the instru - mental limit of detection based on a peak- to - noise ratio of Breast Cancer Res Treat 123 3 was 0.01 –0.03 ng/mL for tetra - through hepta - chlorobi - phenyls, using 1– 1.5 mL plasma. As d escribed previously [23],weusedallobservedpositivevaluesofPCBsin analyses, even those reported to be below the limit of detection. We were able to assay archived serum samples for the PCB congeners which are shown in Table 1.Thesecong - eners include those most commonly reported in prior studies, comprised the congeners with highest concentra - tions, or grouped by potential biological activity as previ - ously recommended [24]. Werandomly assigned the order of samples within and across batches and analyzed c ase– control pairs in the same batches to minimize differences due to laboratory drift. The laboratory was blind as to case or control status of the samples. Intra - batch coefficients of variation ranged from 5 % for PCB 180 to 18 % for PCB 101. Total choles terol and total triglycerides were mea - sured enzymatically on the Hitachi 911 analyzer (Roche Diagnostics, Indianapolis, IN) in a lab certi fied by the Centers for Disease Control and Prevention and the National Heart Lung and Blood Institute Lipid Standard i - zation Program. Statistical analysis This report is based on 112 case–control pairs, matched on year of birth, after excluding 2 pairs with insufficient serum for lipid assays, 2 pairs with missing data on body mass index, and 17 pairs where one or more member of the pair was missing informati on on PCB 167, which was not quanti fied in one of the assay batches. In a sensitivity analysis, we imputed missing PCB 167 by matching on case status and date of blood draw and assigning a PCB value for the missing individual based on a random pick of all m atches. Results were similar (associations were of a similar magnitude with overlapping 95 % con fidence intervals and all associations remained statistically signif- icant, de fined as p\0.05) whether or not the individuals with imputed values were included. We present findings only for individuals with non - missing data on all study Table 1 Distribution of PCBs in breast cancer controls and cases Classi fication [24] PCB Controls (N = 117) Cases (N = 123) Difference within matched pairs (case–control, N = 112) Pe rcent[LOD Percentile (mmol/l) Percentile (mmol/l) 25th 50th 75th 25th 50th 75th Mean (mmol/l) p value Estrogenic 101 0.37 0.74 1.07 0.34 0.55 0.92 - 0.06 0.19 89 187 0.38 0.48 0.66 0.38 0.48 0.63 - 0.07 0.30 100 201 0.09 0.23 0.33 0.14 0.23 0.30 - 0.01 0.71 7 1 Anti - estrogenic A. Non - ortho, mono - ortho, dioxin - like 66 0.89 1.34 1.78 0.96 1.47 1.95 0.10 0.17 99 74 0.62 0.89 1.23 0.62 0.86 1.27 0.01 0.54 95 105 0.25 0.40 0.64 0.25 0.43 0.61 - 0.01 0.92 81 118 1.19 1.62 2.02 1.19 1.53 2.11 - 0.03 0.94 100 156 0.11 0. 28 0.42 0.11 0.28 0.39 - 0.03 0.68 64 167 0.08 0.19 0.30 0.08 0.14 0.25 - 0.03 0.12 49 B. Di - ortho, limited dioxin activity 138 1.58 2.05 2.55 1.50 2.02 2.77 - 0.09 0.56 100 170 0.30 0.46 0.66 0.35 0.48 0.63 - 0.01 0.74 97 Phenobarbital, CYP1A, and CYP2B induc ers 99 0.28 0.58 0.80 0.25 0.52 0.86 - 0.04 0.30 83 153 1.88 2.38 3.08 1.86 2.43 3.08 - 0.08 0.40 100 180 0.89 1.21 1.54 0.94 1.21 1.57 0.01 0.63 100 183 0.13 0.23 0.35 0.10 0.23 0.33 - 0.03 0.32 66 203 0.21 0.30 0.42 0.23 0.35 0.47 0.02 0.20 90 203/167 ? 187 0.34 0.43 0.52 0.38 0.50 0.63 0.09 NA NA Variable number of subjects in each group is due to missing data on one or more of the PCB congeners shown. The PCB score (PCB 203/(PCB 187 ? PCB 167)) was based on the best fitting model as described in the text, a nd is provided to describe the distribution of the mixture of signi ficant PCB predictors. p values are not presented forthe PCB score because the score was created after analysis, as described in text. p values shown are for Wilcoxon Signed Rank Test of di fferences (case–control) within age - matched case–control pairs LOD limit of detection Breast Cancer Res Treat 123 variables, as this is a conservative choice based on observed values. We performed data analysis using age matched condi - tional logistic regr ession as in our prior study of DDT and breast cancer in this same population [25]. We began with a full model, entering all PCBs classi fied by Wolff et al. [24]. as potentially relevant to human health: Group 1 consisted of congeners detected in the CHDS s erum sam - ples that were considered to be potentially estrogenic and persistent (PCB 101,187, 201). Group 2 consisted of congeners detected in the CHDS serum samples that were considered to be potentially antiestrogenic, immunotoxic, dioxin - like: Group 2A which are non - ortho or mono - ortho in their structure (PCB 66, 74,105,118,156,167) and Group 2B which are di - ortho and have more limited dioxin - like activity (PCB 138 and 170). Group 3 consisted of pheno - barbital, CYP1A and CYP2B inducers (PCB 99, 153,180, 183, 203). Congeners that had individual p[0.20 were tested for removal as a group, based on a likelihood ratio test (p \0.15 as the criterion). At the next step, we used a more stringent criterion, eliminating remaining PCB terms with individual signi fican ce probabilities [0.05. In addi - tion to using the likelihood ratio test to test the hypothesis that the coefficients for these terms were each 0 using the criterion, p\0.15, we also examined the sign and size of coefficients of remaining predictors before a nd after elim - ination to rule out major confounding by the eliminated predictors. Our goal was to identify the minimal number of PCBs that predicted risk. Once the best PCB model was identi fied, we examined whether further adjustment for blood lipids (tota l cholesterol, total triglycerides), parity, year of blood draw, body mass index (lower tertile, upper tertile vs. middle tertile as the reference category) and breast feeding following the current pregnancy altered PCB associations with breast cancer. To describe the net effect of PCB exposure on breast cancer, we constructed a post - hoc score that consisted of the ratio of the sum of PCB congener(s) associated with higher risk of breast cancer to the sum PCB congeners associated with lower risk as in a pre vious report on health effects of PCB exposure in this population [26]. We then examined the variation in the post - hoc score and described its association with breast cancer in this population. Results Table 1 shows the distribution of PCB congeners and a post - hoc PCB score based on the final model shown in Table 2. In this study population, PCB congener concen - trations and the distribution of the PCB score were highly variable. The comparisons between cases and controls in Table 1 show no remarkable, stati stically signi ficant associations between single PCB congeners and breast cancer risk, in the absence of control for other congeners. This observation suggests that confounding would play a role in any associations observed in multivariate models. There wer e no associations between the sum of total PCBs or with PCB groups (groups shown in Table 1)and risk of breast cancer (data not shown). Table 2 shows results of multivariate conditional logistic regression models for individual PCB congeners. PCB 203 is t he only congener with a consistent and statistically signi ficant positive coefficient, indicating that it was associated with increased risk of breast cancer (Table 2, both models). In contrast, PCB 167 and PCB 187 were inversely associated with risk of brea st cancer (Table 2, both models). To check for dose response, we also estimated associations for these PCBs by quartile shown in Table 3. Results were largely consistent with a monotonic trend for each congener. Table 2 Associations of individual PCB conge ners with breast can - cer diagnosed before 50 years of age Classi fication [24] PCB Model with all PCBs Final model Coefficient p value Coefficient p value Estrogenic 101 0.39 0.41 187 - 4.18 0.03 - 2.07 0.02 201 - 2.70 0.23 Anti - estrogenic A. Non - ortho, mono - o rtho, dioxin - like 66 0.11 0.61 74 0.36 0.53 105 1.37 0.19 118 - 0.82 0.07 156 - 0.42 0.61 167 - 2.50 0.17 - 2.88 0.03 B. Di ortho, limited dioxin activity 138 0.10 0.87 170 - 1.45 0.34 Phenobarbital, CYP1A, and CYP2B inducers 99 - 0.45 0.56 153 0.10 0.91 180 1. 26 0.28 183 1.15 0.48 203 6.17 0.01 4.36 0.001 In the column labeled, ‘‘Model with all PCBs’’, congeners were entered into a single model as the first step in model selection. Congeners PCB 203, 187, 167 118, and 105 were initially retained based on the cri terion of p \0.20. PCB 118 and 105 were then removed either one at a time or together. They were deleted from the final model by applying the likelihood ratio test where v2 = 3.81, 2df, p = 0.15 for the test to retain both PCB 105 and PCB 118 in the model. B oth were tested for removal simultaneously because deleting either PCB 118 or PCB 105 alone greatly affected the size of coef- ficient for the other. There were N = 112 age - matched case–control pairs for all models shown and all models tested. Coefficients a re reported per 1 mmol/l Breast Cancer Res Treat 123 Based on the results in Tables 2 and 3, a PCB score was constructed as the ratio of PCB 203 (positively related to risk) to the sum of PCBs 167 and 187 (each negatively related to risk). The PCB score wa s highly variable in the cohort, ranging from 8 % PCB 203 (compared to the sum of PCBs 187 and 167) to 131 % PCB 203. Overall for cases and controls combined, the median of the 4th quartile (0.67) was 2.3 times higher than the median of the 1st quartile (0 .30). Among controls, the median of the 4th quartile of the PCB score (0.61) was 2.5 times higher than the median of the 1st quartile of the PCB score (0.25). Among cases, the median of the 4th quartile of the PCB score (0.71) was 2.2 times higher than the median of the 1st quartile of the PCB score (0.32). Table 4 shows the net effect of exposure based on the proportion of PCB 203 compared to the sum of PCBs 167 and 187. Women in the top 25 % of the PCB score had nearly three times the risk of breast cance r as women in the bottom 25 % of the PCB score. Adjustment for lipids or other breast cancer risk factors had little effect on thisresult (Table 4), nor did adjustment for the denominator of the PCB score or adjustment for p,p0 - DDT, o,p0 - DDT, and p,p0 - DDE (data not shown). Figure 1 shows the actual distribution of the within - pair differences for the PCB score for cases versus controls in this study sample. In the majority of case–control pairs (62 %), the PCB score was higher among the woman who subsequent ly developed breast cancer. Pairs where the case had a higher PCB score also showed greater differences on the PCB score than pairs where the control had a higher score (seen in Fig. 1; compare the right side of the Y - axis (center axis) which shows pairs w here cases within the pair had a higher PCB score to the left side of the Y - axis which shows pairs where controls had a higher PCB score). Fig - ure 1 is consistent with the modeling results shown in Tables 2, 3 and 4. Discussion The net effect of PCB expos ure in this study population was nearly a threefold increase in breast cancer risk among women who had a higher proportion of PCB 203 in relation to the sum of PCB 167 and PCB 187 (75th percentile vs. 25th percentile). These results are novel, but not inco n - sistent with the prior literature on PCBs and breast cancer. Three comprehensive reviews concluded previously that human studies of PCBs and breast cancer, which measured exposure in midlife, had variable findings [6–8]. Most prior studies reported prima rily on total PCBs, which are largely determined by the PCBs found in highest concentration in humans (PCB 153, PCB 138, PCB 118, and PCB 180). We too found no associations for total PCBs, high Table 3 Associations of PCB 167, 187, and 203 with breast canc er diagnosed before 50 years of age PCB Quartile Odds ratio (95 % CI) p value for linear trend 167 Q1 1.00 \0.04 Q2 1.09 (0.48, 2.47) Q3 0.70 (0.27, 1.78) Q4 0.24** (0.07, 0.79) 187 Q1 1.00 \0.02 Q2 0.94 (0.41, 2.17) Q3 0.92 (0.36, 2.38) Q4 0.35* (0.11, 1. 14) 203 Q1 1.00 \0.001 Q2 1.21 (0.46, 3.18) Q3 2.89** (0.98, 8.55) Q4 6.34(1.85, 21.73) Each of the three PCBs is coded as quartiles, based on the distribution in controls. Quartile 1 is the reference category and quartiles 2, 3, 4 are entered as dummy variables for each PCB shown. Associations shown are based on a single model where quartile terms for all PCBs are entered. P value for trend is estimated from a linear model where all three PCBs are entered as continuous variables. N = 112 case– control pairs CI con fidence interval, p signi ficance probability * p B 0.10 ** p B 0.05 p B 0.01 Table 4 Estimated net effects of PCB exposure on risk of breast cancer before 50 years of age Level of adjustment Quartile of PCB scorea Odds ratio (95 % CI) Unadjusted Q1 1.00 Q2 1.26 (0.53, 3.00) Q3 1.52 (0.64, 3.62) Q4 3.01 (1.34, 6.78) Cholesterol and trigl ycerides Q1 1.00 Q2 1.23 (0.53, 3.07) Q3 1.53 (0.64, 3.68) Q4 3.09 (1.34, 7.16) Cholesterol, triglycerides, race, parity, lactation, body mass index, and year of blood sampling Q1 1.00 Q2 1.36 (0.53, 3.52) Q3 1.78 (0.70, 4.55) Q4 2.81(1.11, 7.09) CI con fid ence interval a A post - hoc PCB score was de fined to describe the net effect of PCB exposure in this study sample (described in text). PCB 203 was associated with increased risk, while PCBs 167 and 187 were asso - ciated with decreased risk (see final model, T able 2). Therefore, the PCB score was de fined as the proportion of PCB 203 relative to the sum of PCBs 167 and 187: PCB 203/(PCB 167 ? PCB 187). N = 112 age - matched case–control pairs for all models shown Breast Cancer Res Treat 123 concentration PCBs or su ms of PCBs in functional groupings previously proposed by Wolff et al. [24]. No prior studies reported on the independent contribution of the three lower concentration congeners that predicted breast cancer in this study. It is notable that prior studies d id not measure exposure in young women, during windows of susceptibility during early life, when the breast might be more susceptible to endocrine disruption, including in utero, puberty, preg - nancy, or the postpartum [1–3]. Our ability to directly measur e exposure during the early postpartum is a partic - ular strength of this study. A recent meta - analysis of pregnancy- associated breast cancer outcomes found that women diagnosed with breast cancer in pregnancy, and particularly women diagnosed postpartum, had poorer survival [4], providing further evidence supporting the hypothesis that pregnancy and the postpartum period are vulnerable periods for the breast. As PCBs are highly persistent, it is likely that post - partum levels also re flect pregnancy levels, as suggested by one longitudinal study that reported high correspondence between early postpartum levels and levels across all three trimesters of pregnancy [15]. As women were young at the time of blood collection, it is also possible that the early post partum levels of PCBs re flect exposure even prior to pregnancy, possibly during puberty, as well. This might explain the strength of the association observed for PCB 203, a higher chlorinated compound, as compounds with this structure tend to have longer ha lf- lives [14]. Other strengths of this study include prospective assessment of exposure an average of 17 years before diagnosis, simultaneous consideration of individual PCB congener effects, and the opportunity to observe a popu - lation during active expo sure because blood samples were obtained before PCBs were restricted. Our focus on breast cancer at a young age is an addi - tional strength. Molecular studies strongly suggest that pre - menopausal breast cancer may not share the same features or risk facto rs as breast cancer diagnosed in middle age and older [27, 28]. Our findings could lead to better under- standing about etiology, prevention, and treatment of early breast cancer, if the mechanisms for the associations we observed can be validated and inves tigated by experimental toxicology and molecular studies. Our choice to estimate the net effect of exposure to observed PCB mixtures found in our study participants is an additional strength. While our approach will likely be improved upon as the methods for analyzing mixed expo - sures advances; here, we applied an empirical approach to describe more than individual congener associations. It is of interest that the protective associations for PCBs 187 and 167 did not overcome the stronger, deleterious asso - ciation observed for PCB 203. There are several specula - tiveexplanations for this finding, the first being that PCB 203 is a particularly strong risk factor, as evidenced by its point estimate. Alternatively, as the higher chlorinated PCBs are eliminated more slowly, it is possible that post partum levels of PCB 203 more accurately re flect exposure even earlier in life, including accumulations in utero, childhood and during puberty, periods of susceptibility for the breast in addition to pregnancy and post partum. Limitations of our study include the possibility of unmeasured confounding by other exposures. In particular, we were unable to measure dioxin exposure or activity, raising the possibility that this or other unmeasured con founders could have mask ed or accounted for the associ - ations we report here. However, as we observed dose response, unmeasured confounding would have to follow the same pattern, making this alternative explanation of our findings less likely. It is also possible that host factor s that in fluence the metabolism or selective excretion of various PCB congeners underlie the associations we observed. We used early postpartum samples to save valuable timed serum samples during pregnancy for other studies in the cohort. However, prior stu dies conducted in serum samples of the same age found good correspondence of these per - sistent organochlorines across all trimesters and the early postpartum [15], suggesting that postpartum PCB levels 0 10 20 30 40 50 60 70 80 90 100

- 1.05 - 0.75 - 0.45 - 0 .15 0.15 0.45 0.75 1.05 Intra - pair difference for the PCB score (case - control) Cumulative Percent Fig. 1 Cumulative distribution of case–control differences for the PCB score (N = 112 age - matched case control pairs). Each point represents one case–control pair. The points on the right side of the y - axis (center axis) arepositive values that represent pairs where the woman who developed breast cancer had a higher PCB score postpartum than her matched control. In a majority of pairs (62 %), the woman who su bsequently developed breast cancer had a higher PCB score. The differential for the PCB score was also greater for pairs where the case had a higher score than her matched control (compare points on the right of the y - axis to points on the left of the y- a xis) Breast Cancer Res Treat 123 may also re flect pregnancy exposures. Still our study can - not establish the age or developmental period when PCB exposure was acquired, other than establishing that expo - sure preceded the mean age of blood collection (age 26 years). Storage of serum samples is unlikely to have biased study results, as all samples were similarly stored. Randomization of samples within and between batches and inclusion of controls and cases in the same batches mini - mized inter- and intra - bat ch laboratory error. Interpretation is limited by a lack of understanding about the potential mechanism for PCB associations observed. The direct association between PCB 203 and early breast cancer was sizable and signi ficant. However, the mode of action fo r PCB 203, classi fied as a phenobarbital (PB) inducer [24], is unknown. A PubMed search for ‘‘PCB 203’’ returned no citations, compared to 589 citations for ‘‘PCB 153,’’ for example. The Agency for Toxic Sub - stances and Disease Registry report on the toxic effects of PCBs makes no speci fic mention of PCB 203 effects [29]. PCB 187 has been classified as potentially estrogenic [24], but it is unclear how postpartum or pregnancy exposure might be associated with a lower risk of breast cancer. This study provides little information regarding the validity of the classi fication of PCB 187 as estrogenic. In our previous report on the relation of prenatal PCB expo - sure to time to pregnancy in daughters in this same cohort [26], we found that a longer time to pregnancy in daughters was associated with prenatal exposure to PCB 187. The signi ficant associations observed for breast cancer in mothers and time to pregnancy in daughters following prenatal exposure to PCB 187 deserves additional study, perhaps in experimental m odels or in vitro systems. It could be of interest to characterize PCB 187 effects in the case of low versus high endogenous levels of estrogen and during pregnancy and postpartum in particular. We were unable to investigate gene/PCB interactions in this s tudy. The absence of associations for some of the PCBs investigated might be explained by failure to identify susceptible sub - populations of women. Previously, asso - ciations were more consistently observed in sub - popula - tions characterized by variant all eles for enzymes that metabolize PCBs (Cytochrome P4501A1 variants) [10– 13]. We were also unable to characterize the receptor status of the breast tumors in our study. We cannot account for lactation in subsequent pregnancies. However, breast feeding follo wing the current pregnancy did not predict breast cancer in this sample and was not a confounder of PCB associations. There was no correlation between breast feeding following the observed pregnancy and PCB 203, 187, or 167. These findings may be explained by the low frequency of long - term breast feeding in our cohort. Rates of lactation in this sample were low (34 %) and among those who did breast feed, most (60 %) breast fed for \4 months [25]. We suggest that it is unlikely that lacta - tion in subsequent pregnancies explains our results, as lactation behavior is highly correlated among pregnancies [30], but this remains a possibility. The variabledistribution of PCB congeners observed in study subjects is to be expected as the distribution of congeners depends on source of exposure which is in flu - enced by the chemical structure of each congener. Expo - sure also depends on the fate of the congener in the ecosystem which ultimately forms the source of human exposure and on the individual respo nse to the exposure. All these factors contribute differently to the measured serum level [31]. In a previous report on in utero PCB exposure and daughter’s time to pregnancy, we also found considerable variability in the mixture of PCB congeners in CHDS m others [26]. In the US, total PCB levels in adipose tissue declined steadily after 1972 when restrictions were implemented. A decline in PCBs was also observed in archived blood samples in Norway during the same period [32] and in human adipose tissue in t he United States [33]. However, secular trends in the mix of congeners are unknown [33]. As the fate of individual congeners in the environment depends on their structure, environmental topography, and climate, and because individual charac - teristics may determine routes of exposure, and metabolic fate, it is unlikely that trends for individual congeners are the same over time, or within individuals, or across geo - graphic areas [14]. There is little human data on this topic, but results of repeated blood sampling in a Danish cohort provides limited support for the concept that congener proportions vary over time: over a 5 - year period (1976– 1978 vs. 1981– 1983) median concentration of total PCBs declined 11 %, but median concentration of PCB 118 declined 34 %, PCB 180 declined 4 %, and PCB 153 declined 9 %.(adapted from Hoyer, et al., Table 1, p. 179) [34]. If congener mixture is the underlying risk factor, then we might expect different results for epidemiological studies, depending on place, time, age, and other individual characteristics that might alter external and internal dose to these compounds. Given the variability of congener mixtures observed in our cohort, we speculate that an underlying host factor related to metabolism of these compounds might c ontribute to the PCB associations with breast cancer that we have observed in this study. These data do not allow us to determine whether PCB exposure would be necessary to trigger an effect, or whether some host factor might be sufficient to increase breas t cancer risk. Our analysis does indicate that PCB associations observed in this study are independent of DDT associations previously observed in this cohort [25]. Mechanistic studies in experimental models, or in vitro are likely to be very important to e xplaining the associations we report here, and for Breast Cancer Res Treat 123 explaining the human breast cancer associations previously reported for CYP1A1 polymorphisms in relation to PCB exposure in middle - aged women [10– 13]. In summary, in this study, the mixture of PCB congeners predicted the estimated effect of PCB exposure on risk of breast cancer. Overall, women in this study showeda variable distribution for the three PCB congeners that predicted breast cancer. Women with a high proportion of PCB 203 (top 25 % of the study population) relative to PCBs 167 and 187 had a nearly threefold increase in subsequent risk of breast cancer, compared to women with a lower proportion of PCB 203 (bottom 25 % of the study population). The relation of PCB exposur e to breast cancer might be clari fied by additional laboratory, experimental and human population studies that account for timing of exposure in relation to windows of susceptibility for the breast and for concomitant host factors. It is likely to be partic ularly important to studycongener mixtures and individual response to multiple exposures. It remains unclear whether individual differences in exposure, response to exposure, or both explain risk patterns observed. Acknowledgments This publication was mad e possible by the Breast Cancer and the Environment Research Program (BCERP) award number (U01 ES/CA 019471 to B.A.C. and M.B.T.), from the National Institute of Environmental Health Sciences (NIEHS) and the National Cancer Institute (NCI), NIH, DHHS; the National Cancer Institute award number (R01 CA72919 to B.A.C); and The National Institute of Child Health and Human Development award numbers (N01 HD 6 3258 and N01 HD 1 3334 to B.A.C.). Its contents are solely the responsibility of the authors and do not necessarily repre - sent the official views of the NIEHS or NCI, NICHD or the National Institutes of Health. We also gratefully acknowledge support of the Avon Foundation for this research, and our collaborators Mary Wolff who performed serum assays for this study, Robert Sholtz and Nikilou Krigbaum who prepared data files, Linda S. Birnbaum who provided comments on an earlier version of this manuscript, Barbara J. van den Berg, Roberta Christianson the late Frank Oechsli and the late Jacob Yerushalmy, Child H ealth and Development Studies founder, who together created and preserved the Child Health and Development Studies cohort. We also thank the National Institute of Child Health and Human Development, both current and past directors, Alan Guttmacher and Dway ne Alexander, and our project officer, Gilman Grave for supporting the maintenance of the CHDS cohort. The collection of cancer incidence data used in this study was supported by the California Department of Public Health as part of the statewide cancer rep orting program mandated by California Health and Safety Code Section 103885; the National Cancer Institute’s Surveillance, Epidemiology and End Results Program under contract N01 - PC35136 awarded to the Northern California Cancer Center, contract N01 - PC - 35 139 awarded to the University of Southern California, and contract N02 - PC - 15105 awarded to the Public Health Institute; and the Cen - ters for Disease Control and Prevention’s National Program of Cancer Registries, under agreement #U55/CCR921930 - 02 awarded to the Public Health Institute. The ideas and opinions expressed here in are those of the author(s) and endorsement by the State of California, Department of Public Health, the National Cancer Institute, and the Centers for Disease Control and Prevention o r their contractors and subcontractors is not intended nor should be inferred. Con flict of interest The authors declare that they have no conflict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncom mercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References 1. Fenton SE (2006) Endocrine - disrupting compounds and mam - mary gland development: early exposure and later life conse - quences. Endocrinology 147(6):s18–s24 2. 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Cancer Causes Control 11(2):177– 184 Breast Cancer Res Treat 123Number of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1975 11.1 - 5.6 1976 11.2 - 5.7 1977 11.2 - 5.8 1978 11.9 - 5.9 1979 12.5 - 5.9 1980 12.6 - 6.2 1981 13.6 - 6.2 1982 13.4 - 6.6 1983 14.0 - 6.7 1984 15.2 - 6.8 1985 15.5 - 7.1 1986 15.9 - 7.3 1987 16.7 - 7.3 1988 17.2 - 7.5 1989 17.4 - 7.8Number of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1990 18.5 - 7.9 1991 18.8 - 8.2 1992 18.6 18.5 8.2 1993 18.9 18.5 8.2 1994 20.0 19.6 8.6 1995 20.0 19.7 8.7 1996 19.4 19.4 8.8 1997 20.0 19.5 8.9 1998 19.6 19.3 8.7 1999 19.9 19.8 8.3 2000 19.7 19.4 8.2 2001 20.0 19.7 7.9 2002 20.2 19.7 7.7 2003 20.7 20.2 7.4 2004 21.4 20.8 7.1Number of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  2005 20.8 20.2 6.9 2006 20.4 20.0 6.7 2007 21.1 20.4 6.6 2008 20.7 20.2 6.4 2009 20.7 20.4 6.3 2010 21.1 20.6 6.1 2011 19.6 19.2 -Journal of Environmental and Public Health

Volume 2012 (2012), Article ID 258981, 15 pages http://dx.doi.org/10.1155/2012/258981 

### Review Article 

## Plasma Levels of Polychlorinated Biphenyls, Non- Hodgkin Lymphoma, and Causation Michael D. Freeman 1 and Sean S. Kohles 2 1 Department of Public Health and Preventive Medicine, Oregon Health & Science University

School of Medicine, 1234 SW 18th Avenue, Suite 102, Portland, OR 97205, USA 2Regenerative Bioengineering Laboratory, Department of Mechanical & Materials Engineering, Portland State University, 1930 SW 4th Avenue, Suite 4 00, Portland, OR 97201, USA Received 21 June 2011; Accepted 9 November 2011 Academic Editor: David O. Carpenter Cop yright © 2012 Michael D. Freeman and Sean S. Kohles. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 

### Abstract Polychlorinated biphenyls (PCBs) are sy nthetic chlorinated hydrocarbons that have extensively

p olluted the environment and bioaccumulated in the food chain. PCBs have been deemed to be probable carcinogens by the Environmental Protection Agenc y, and exposure to high levels of PCBs has been consistently linked to increased risk of nonHodgkin lymphoma (NHL). In the present article we present a forensic epidemiologic evaluation of the causal relationship between NHL and elevated PCB levels via application of the Bradford- Hill criteria. Included in the evaluation is a meta- analysis of the results of previously

published case - control studies in order to assess the strength of association between NHL and PCBs, resulting in an odds ratio in which the lowest percentile PCB concentration (quartile, quintile, or tertile) has been compared with the highest percentile concentration in the study groups. The weight- adjusted odds ratio for all PCB congeners was 1.43 with a 95% confidence interval of 1.31 to 1.55, indicating a statistically

s ignificant causal association with NHL. Because of the lack of an unexposed comparison group, a rationale for the use of a less than 2.0 relative risk causal contribution threshold is presented herein, including an ecologic analysis of NHLincidence and PCB accumulation (as measured by s ales volume) over time. The overall results presented here indicate a strong general causal association between NHL and PCB exposure. 

### 1. Introduction Polychlorinated biphenyls (PCBs) are a class of commercially-produced organochlorines known as chlorinated hydrocarbons [ 1 ]. PCBs are nonflammable, chemically stable, have a high boiling point, and are nearly insoluble in water. In addition, they are resistant to the effects of

oxidation, acids, bases, and other chemicals [ 2 , 3 ]. A PCB mo lecule consists of a pair of joined 6 - carbon rings, with chlorine(s) attached or “substituted” at any one of the free 10 - carbon positions. There are 209 possible chlorine arrangements, which are called congeners. It is the number of the chlorines and where they are attached that determine the congener’s properties. A congener’s two - carbon rings can be twisted, relative to each other, or they c an be aligned in the same plane (coplanar). Coplanar carbon ring alignment occurs when the chlorine(s) are attached to the carbons closest to the link between the two rings, called the ortho position. Coplanar congeners exhibit dioxin- like properties [ 4 , 5 ]. For approximately

5 0 ye ars (beginning in 1929), PCBs were manufactured in the United States (USA) and were used in numerous industrial and commercial applications in the form of mixtures called Aroclors [ 6 ]. One manufacturer (Monsanto Company) has produced ~99% of all the PCBs used in the USA [ 2 ]. Worldwide, Monsanto has produced between 39 and 48 percent of all PCBs. Widespread industrial use of PCBs, combined with improper disposal practices, has led to the introduction of PCBs into the environment, where these chemicals are found in all environmental me dia, including air, water, and soil. Based on mounting evidence that PCBs were accumulating and persisting in the environment and that this accumulation was causing adverse health effects in humans and animals, PCB production in the USA was halted in the late 1970s [ 1 ]. PCBs are highly

s oluble in lipids, and as such, are absorbed by f ish and other animals, leading to considerable bioaccumulation in the food chain. PCBs are ty pically absorbed into the human body

through ingestion, inhalati on, and/or dermal exposure [ 1 , 7

–9 ]. Bioaccumulation in humans and animals occurs when PCBs are absorbed into the body at a rate greater than the rate at which they are metabolized and excreted from the body . Detectable levels of PCBs have been found in humans in adipose (fat) tissue, breast milk, hair, and serum lipids [ 3 , 10 ]. Consumption of contaminated food is the major source of PCB exposure for the general United States population that is not occupationally exposed to PCBs [ 1 , 2 , 8 , 10 , 11 ]. Unsurprisingly, populations that are at particular risk to high exposure to PCBs include people who consume sport- caught fish and other contaminated foods, workers occupationally exposed to PCBs, individuals residing near hazardous waste sites containing PCBs, and nursing infants [ 7 , 8 , 10 , 12 ].PCBs are deemed to be probable carcinogens by the Environmental Protection Agenc y based on the results of both animal and human studies [ 1 ]. A number of animal studies have demonstrated a direct dose -response relationship between PCB levels and liver tumor occurrence [ 13

– 16 ]. In humans, there is also good evidence of an association between increased cancer rates and PCBs. A num ber of epidemiologic studies have indicated increased rates of liver and biliary cancer [ 17 ], breast cancer [ 18 ], skin cancer [ 19 ], and Non- Hodgkin Lymphoma (NHL), among other cancers [ 20 ]. PCBs appear to have a number of toxic qualities that potentially explain these observed associations, including dioxin- like characteristics of some congeners (118, 156, and 169) [ 5 ], the ability

to mimic hormones for others [ 11 ], and neuro - and immunotoxicity as well [ 21

–23 ]. This last quality (immunotoxicity) is helpful in explaining the observed association between elevation of certain congener titers (118, 138, 153, 170, and 180) and the increased incidence of NHL, as immune system depression is considered to be one of the strongest risk factors for NHL [ 24 , 25 ]. Immunotoxicity i s a characteristic shared by other organochlorines as well, including dioxins and chemicals found in pesticide s and herbicides such as hexachlorobenzene, heptachlor, chlordane, and others [ 26 ], which have also been found to be associated with increased risk of NHL [ 27 ]. It is most likel y the quality

o f immunotoxicity

that links both PCBs and non- PCB organochlorines to increased incidence of NHL, and when both are present, each contribute to the risk of NHL independently

[ 26 ]. NHL includes all cancers of ly mphoid tissues except Hodgkin’s disease, a malignancy o f the ly mph nodes [ 28 ]. The incidence of NHL in many p arts of the world is rising more rapidly than the incidence of virtually all other human cancers [ 29 , 30 ]. In the US, the incidence of NHL increased at an average of 3.6% per ye ar from 1975

– 1991 and continued to rise over the period 1991

–2005, albeit at a slower rate (~0.5% annually) [ 31 , 32 ]. It is widely

accepted that changes in diagnostic practices and known risk factors such as age, autoimmune disease incidence, and prevalence of immune - suppressing infections are insufficient to explain the emerging “epidemic” of NHL observed in most of the world [ 33 , 34 ]. Additionally, it is hy pothesized that exposures shared by many populations worldwide are the most likely explanation for the steep increases in NHL incidence [ 31 , 33

–35 ], and that this exposure is likely immunotoxic or immunosuppressive [ 36 ]. Thus, exposure to PCBs and other persistent organic pollutants provides a reasonably

p lausible explanation, at least in part, for the rise in NHL incidence during the latter half of the 20th century . The underlying physiologic mechanisms for the development of NHL secondary to PCB exposure are not fully known. It is well established that the dioxin- like congeners can bind to and activate the aryl hydrocarbon receptor (AhR), a normally inactive transcription factor [ 37 ]. The overactivation of the AhR can induce enzymes that produce cy totoxic metabolites or otherwise adversely affect cellular metabolism [ 38 ]. The degree to which a particular coplanar dioxin- like congener can act on the AhR is measured in Toxic Equivalents (TEQ), a comparison with a standard set to the highly toxic dioxin- likecompound 2,3,7,8 - Tetrachlorodibenzo -p - dioxin (TCDD) [ 39 ]. Less is known about the

noncoplanar nondioxin - like congeners, however, they appear to affect immune function by direct cellular effects, including inhibition of leukoc ytic phagocy tosis, among other actions [ 22 ]. Although there are some differences in the specific congeners measured and variations in the PCB levels associated with increased risk of NHL, the literature generally supports, via both case - control and cohort studies, the concept that populations with higher levels of exposure to certain PCB congener and with higher body burdens of these same congeners are at increased risk for NHL. Multiple representative publications describe groups of previously identified cases of NHL from various registries and clinical investigations and compared them with age and gender- matched controls, resulting in quantitative descriptions of relative influence of percentile groupings of blood plasma PCB on NHL risk via conditional logistic regression (Table 1 ) [ 26 , 40

–48 ]. An additional study [ 49 ], analyzed adipose tissue in a postmortem assessment. Rothman et al. [ 40 ], Engel et al. [ 41 , 48 ], all used prediagnosis blood samples (not subject to weight loss bias). Additionally, three landmark exposure studies investigated the association between environmental levels of various Aroclor products and mortality rates from NHL (Table 2 ) [ 50

–52 ]. These studies collectively have provided a foundation for a reasonable conclusion that a causal relationship exists between high levels of PCBs, both in the environment and in the body and increased risk of NHL.

Table 1: Summary

o f published case - control study populations examining risk of NHL and PCB blood content including those meta- analyzed in the present study. S ummed or total PCB metrics shown here.

Table 2: Summary

o f published cohort studies examining mortality f rom NHL as associated with PCB exposure. Note the cohort described in Prince et al., [ 50 ] were included in the Prince et al., [ 51 ] study . Here, the standardized mortality ratio (SMR) was calculated as an indirect adjustment method where the observed number of events (deaths) in each occupational cohort is com pared with the num

ber of expected events (based on a “standard” rate). A few authors have reported subgroups of NHL that are linked to PCB exposure, including an association between diffuse large cell ly mphoma and congener 118 and T cell ly mphoma and congener 180 [ 42 ]. Hardell reported the highest observed associationin their cohort between elevated PCB burdens and low- grade B - cell lymphomas, the most

prevalent category

o f NHL subty

pes [ 43 ]. DeRoos found an association between congeners 180 and 187 and diffuse but not follicular NHL, whereas Morton reported an association between follicular NHL and congener 18 0, as well as marginal zone NHL subty pe [ 54 ]. Given a reasonable basis for a conclusion of general (population-based) causation, the question arises of how to determine, in an individual case of NHL in which elevated blood and/or adipose levels of total or individual PCB congeners have been observed, if the relationship between the two is causal. Far more has been written in epidemiology about the evaluation of general (population) c ausation than specific (individual) causation. Epidemiologic methods are used for the investigation of specific causation in both short acting exposure/outcome situations (i.e., outbreak/injury investigation) and in settings in which disease onset is latent, sometimes for decades, following exposure, such as with exposure to environmental toxicological or radiological hazards. The discipline of forensic epidemiology is directed at addressing, often for presentation in legal settings, the specific causal correlation between a suspected hazard and a disease or injury outcome [ 55

–57 ]. The ultimate goal of the inve stigation is to answer the “but- for” question, which is “but- for the exposure to the hazard, would the individual still have the disease or injury? ” [ 58 ]. The forensic epidemiologic approach to answering this question is accomplished by

f irst assessing the plausibility o f a causal relationship via application of the Bradford- Hill criteria and then estimating the attributable risk percent or probability

o f causation (PC) that quantifi es the probability that, but for the exposure, the disease or injury outcome would not have occurred [ 59 ]. Generally, if the PC exceeds 50% then the hazardous exposure is considered most probably c ausally related to the disease or injury

outcome [ 60 ]. PC is derived from comparative epidemiologic data that adequately

represent the relative risk (RR) of disease or injury b etween hazard- exposed populations most me aningfully s imilar to the individual versus nonhazard- exposed populations most meaningfully s imilar to the individual. A PC of >50% is the equivalent of an RR >2.0 and implies that the cause of disease or injury in an individual randomly selected from the exposed population is the hazard of interest more often than not. This approach is relatively

straightforward for evaluating the causal relationship between, for example, cigarette smoking and pancreatic cancer, as there are clearly defined and easily identified exposed (smoking) and unexposed (nonsmoking, not exposed to second-hand smoke) populations. For the evaluation of PCB exposure and NHL, the issue of causation is more complicated, as the ubiquitous nature of PCBs in the environment means that there are no truly unexposed populations. In com bination with the likely

multifactorial etiology

o f NHL (viral infections, immune system depression, autoimmune conditions, and genetic anomalies are all thought to play a role), it is much easier to conclude that the prominence of a single “suspect” hazard- like elevated serum levels of PCBs serves to contribute to the cause of an individual’s NHL such that the hy pothetical complete subtraction of the hazard would likely result in the individual not developing the disease(the factor is necessary for the development of the condition). Conversely, the

development of NHL most likely requires a number of components along with the suspect hazard, any

num ber of which may also be necessary in order for the disease to manifest. In the present endeavor, we propose a methodologic framework for the evaluation of specific causation of NHL associated with PCB exposure that takes into account the pervasive and persistent nature of PCBs in the environment and the difficulties with setting an absolute threshold for specific causation. To accomplish this task, we present a meta- analysis of the previously published case - control studies that have examined the risk of NHL relative to PCB titer by c ongener. Additionally, we discuss a rationale for the use of an adjusted relative risk or probability o f causation threshold for concluding that a causally

c ontributory

relationship is more probably than not present in an individual with NHL and elevated PCB titers. 

### 2. Methods 2.1. Application of Hill Criteria for Causal Assessment For the purposes of the present study, a causal relationship is defined as when an exposure is found to have served as an antecedent event or condition that was necessary for the occurrence of a specific disease or injury at the moment that it occurred, given that other conditions are fixed [ 61 ]. That is to say, the cause of the disease or injury i s an event or condition that preceded the disease or injury and without which the disease or injury would not have occurred. Forensic applications o f epidemiology that address the evaluation of causation generally f ollow the criteria set forth by S ir Austin Bradford- Hill in 1965 [ 55 , 56 , 62

–66 ]. Hill’s nine criteria, in the order in which they w ere given in the original publication, are briefly as follows.(1) Strength of association : strength of association is the most important determinant of both general and specific causation and quantified by

RR, in that the larger the ratio between the incidence of the condition in the exposed group versuses the incidence in the unexposed group, the greater the probability that the relationship is causal. As described previously, an RR >2.0 is the equivalent of a PC >50%, meaning that it is more probable than not that the suspected causal relationship is true.(2) Consistency : the repetitive observation of a relationship in different circumstances strengthens the causal inference.(3) Specificity : the degree to which a suspected causal factor is associated with a particular outcome or population.(4) Temporality : the potential causal factor must precede the outcome it is assumed to affect, and the outcome cannot either occur before it is physiologically feasible or after too great of a latency p eriod. Temporality

i s the one factor that mu st always be present in general and specific causation in order to conclude that a cause and effect relationship is present.(5) Biological gradient : the injury outcome increases proportionately

with increasing dose of exposure (also known as dose re sponse).(6) Plausibility : the degree to which the observed association can be explained by known scientific principles.(7) Coherence : a causal conclusion should notfundamentally c ontradict present substantive knowledge; it should “make sense” given current knowledge.(8) Experiment : in some cases, there may be evidence from randomized experiments on animals or humans.(9)Analogy : an analogous exposure and outcome may be translatable to the circumstances of a previously unexplored causal investigation. For appli cation in a forensic setting, these criteria are sometimes mo dified to include cessation (of exposure) as a test-retest criteria and consideration of alternative explanations for the association (such as bias and confounding), in place of the less frequently

useful experiment criterion [ 60 ]. The only criterion that is truly

e ssential for a causal association is temporality, as the outcome must follow the exposure in time. In fact, none of the criteria (with the exception of temporality) are applicable in all circumstanc es [ 67 , 68 ]. The causation criteria can be lumped into three main groups by their utility : (1) those used to evaluate whether there is a reasonably p lausible relationship between outcome and exposure ( consistency, specificity, biological gradient, plausibility, coherence, experiment, and analogy and cessation); (2) temporality ; (3) strength of association (or probability

o f causation). An analysis involving the application of these criteria to the evaluation of the plausibility o f a causal relationship between NHL and elevated PCB congener titers, as well as temporality, i s presented in the results and discussi on sections. The final criterion, strength of association, is discussed below. 2.2. Meta- Analysis of Individual Congeners as a Measure of Strength of Association (Causal Contribution) In order to assess the utility o f PCB congener level as an index of caus al contribution, it was necessary

to meta- analyze data from previously published case - control studies in which the risk of NHL was compared with serum levels of individual congeners through the application of conditional logistic regression. In order to be included in the metaanalysis, the studies needed to have the following characteristics in common: they examined individual PCB congeners identified in plasma samples acquired from cases of NHL (as opposed to groups of congeners), they included age and gender frequencymatched controls, they ranked their results by p ercentile of congener concentration (from lowest to highest tertile, quartile, or quintile), they quantified their results in terms of natural log odds ratios as a measure of NHL risk by c om paring the minimum percentile with the maximum, and multiple strata were available for any included congener. For the meta- analysis, the fixed- effect Mantel - Haenszel (MH) - adjusted odds ratio (ORMH) was calculated as a weighted average ( ) of the natural lo g (ln) odds ratio (OR ) for each study in which the minimum percentile was compared with the highest percentile concentration (Table 3 ). This minimum- maximum comparison was chosen for the metaanalysis in order to summarize the com parison of the extrema. The following calculation was used: ORMH=∑ =1 / × / ∑ =1 / =∑ =1 OR ∑ =1 .(1 )This approach utilized the subpopulations within each study ( , , , and ) as

identified in the two -by- two contingency tables stratified by studies. The confidence interval for the ORMH was determined by f irst calculating the standard error (SE) of the OR estimate (OR ) and given by the following equation [ 69 ]: S E lnO RMH = ⎷ ∑ =1 2 ∑ =1 2+∑ =1 + 2 ∑ =1 ∑ =1 +∑ =1 2 ∑ =1 2 , (2 ) where = + , = + , = × , = × .(3) The meta- analyzed 95% confidence level (CI) was then calculated in the natural logarithmic (ln) scale to match the scale of the OR:[].9 5%CI(lnOR)=lnOR±1.96×SE(lnOR)(4) Table 3: Notation for the calculation of Mantel - Haenszel - adjusted odds ratios from the published case - control studies reporting association of NHL and PCB blood content for specific congeners. Here, “exposed” and “unexposed” populations noted in traditional meta- analyses were replaced with “upper” and “lower” acknowledging the upper and lower percentiles of exposure reported in the literature, respectively . The subscript “ ” indicates each stratum (published congener). A test for homogeneity

was also conducted to assess the application of the fixed- effect mo del applied here to the published ORs. This approach was applied to test whether the population ORs are in fact constant across the different strata [ 70 ]. If the test fails, the ORs can simply be reported as distinct values or be further meta- analyzed using a random- effects model [ 27 , 71 ]. The test for homogeneity evaluates the null hy

pothesis ( ) where the population odds ratios for the tables are assumed statistically i dentical, or equivalently, =OR1=OR2= ⋯ =OR = ⋯ =OR . To perform this test, we calculated the chi - square statistic ( or 2): = 2= =1

− 2.(5) Here the natural logarithm of each estimated OR is determined: =lnOR (6) and used to produce a weighted average ( ) applying the weighting value described in ( 1 ), such that: ∑ = =1 ∑ =1 .(7) The resulting statistic from ( 5 ) has a distribution that is approximately chi - square with g 1 degrees of freedom. A chi - square distribution table was then consulted for each test, producing a value as an assessment of the null hy pothe sis, where <0.05 was considered as a rejection of the null [ 70 ]. An alternative test statistic for assessing homogeneity

is the likelihood ratio test, which is computationally more cumbersome than the statistic applied here [ 72 ]. 2.3. Ecological Analysis In order to explicate the correlation between changes in environmental levels of PCBs and changes in the incidence of NHL an ecologic effect analysis was performed. Data were acquired from the National Institute of Cancer [ 32 ] and compared to PCB production and sales levels [ 6 , 73 ]. Time - dependent accumulation of PCBs was calculated from the annual sales data of the open source materials including heat transfer products, hydraulics/lubricants, miscellaneous industrial products, plasticizers, and petroleum additives. The environmental PCB accumulation data are presented in threeforms: first, only the sales data are plotted over time; second, the sales data are extended in time, assuming a static level of accumulation where the maximum presence of PCB in the environment remains fixed and constant after production ceased; third, the sales data are used as a basis from which to assume a dy namic accumulation of PCB, where environmental levels increase as devices and materials break down, continuously releasing congeners into the environment. This last approach required forecasting the accumulation beyond the end of production following a similar initial growth curve into the future. A poly

nomial curvefit produced a strong phenomenological model representing the time - dependent accumulation in units of kilo - lbs ( 2=0.9772), with time measured in ye ars:PCBAccumulation=16,488.2+1048.9(Time)2.(8) This model was then applied to ye ars beyond the end of the sales period, extending the accumulation data into the future, matching the surveillance period for NHL incidence. Plotted comparisons were made over the actual dates (calendar ye ars) as well as relative time from origination (both in sales and cancer incidence monitoring). The environmental PCB accumulation model was juxtaposed with the incidence over time of a number of major cancer ty pes. A correlation coefficient was calculated for each NHL and non-NHL association, where 2>0.80 was considered a strong statistical correlation. 

### 3. Results 3.1. Assessment of the Hill Criteria for Causation 3.1.1. Plausibility In the present context, plausibility does not refer to Hill’s narrow use of the term as one of the nine causal criteria, in which he referred to the specific biologic action by which an environmental factor caused a disease, but rather to the group of criteria (all but temporality

and strength of association) that answer the question “ can the exposure cause the outcome ?” Interestingly, Hill did not consider the biological plausibility criterion to be particularly

critical to a finding of cause and effect, stating that he was “ convinced this is a feature that we cannot demand” [ 62 ]. This was because of the recognition that an environmental toxin may, in fact, cause a disease by acurrently

unexplicated mechanism that may be described in the future. In examining the plausibility o f a causal relationship between PCB exposure and NHL, there is a substantial amount of published information to rely upon that indicates that the relationship is indeed plausible. Analogy i s strongly

supported for PCB exposure as a cause of NHL, as they

b elong to the same chemical family o f organochlorines as other chemicals that have been associated with NHL in prior epidemiologic studies [ 27 ]. From a biological plausibility

p erspective, NHL is a disease that has been repeatedly shown to be related to compromise of the body ’s immune response [ 24 , 25 ], and there are a number of PCB congeners that have been deemed immunotoxic [ 74 ]. Additionally, thedioxin- like PCB congeners have the ability to bind to the aryl hydrocarbon receptor, a normally inactive transcription factor that when bound can alter genetic transcription. Consistenc y of the relationship is seen with the number of published epidemiologic studies, both case - control and cohort design, of various populations in various settings in which an association between PCB congener titer levels has been linked to NHL risk (Tables 1 and 2 ). 3.1.2. Temporality Because of the ubiquitous nature of PCBs in the environment and the way in which they accumulate over time in the body, as well as the nature of NHL as a disease that has seen its largest increases in the population over the age of 55, aside from the youngest patients with NHL (often those with readily app arent explanations for the disease, such as immunosuppressive infections), temporality i s assumed to be appropriately present in mo st cases of NHL in the presence of high PCB titers. 3.1.3. Strength of Association (Causal Contribution) via Meta-Analysis El even published case - control studies reported on the association between NHL and PCB levels were considered for meta- analysis (Table 1 ). Of these publications, six articles described seven unique populations associating the influence of 10 congeners (28, 99, 118, 138, 153, 156, 170, 180, 183, and 187) on NHL incidence and which were deemed eligible under the meta- analysis inclusion criteria. Point- estimate odds ratio results f or all studied congeners from each of the published case - control studies are described via horizontal forest plot as a summary o f the previous published results (Figure 1 ). These results were then meta- analyzed according to the methods described previously (Figure 2 ). Figure 1: Odds ratios and 95% confidence limits for all PCB blood- level congeners as associated with NHL and reported in six case - control studies.

Figure 2: Meta- analyzed congeners indicating Mantel - Haenszel - adjusted odds ratios and 95% confidence limits for all PCB blood- level congeners with two or more strata including the ORMH for all congeners combined. Congeners with single study analyses are also included (congeners 99, 156, 183, and 187). Statistically s ignificant confidence intervals are indicated (*). Meta- analyzed upper percentile cutoff PCB blood- level values are also included for each congener (ng/g lipid).The weight- adjusted odds ratio (ORMH) for all 10 congeners collectively was 1.43 (95% CI 1.31

– 1.55). Each of the 10 congeners contributed to its own congener- specific metaanalysis as well as toward the all - cong ener ORMH . ORMH results for seven congeners (118, 138, 153, 156, 170, 180, and 187) were statistically s ignificant, whereas the results for three congeners (28, 99, and 183) were not (Figure 2 and Table 4 ). All of the seven congeners with significant meta- analysis results have been previously described as having immunotoxic characteristics, and one (118) is also considered to be dioxin like.

Table 4: List of contributing strata and OR metrics satisf ying the meta- analysis criteria. Results of the Chi - square test for homogeneity ( values and degrees of freedom, DOF) are shown for each meta- analyzed congener. Note that congener 118 is statistically heterogeneous ( <0.025). However, removal of the weighted outlier [ 46 ] indicated homogeneity

in the remaining strata. A combined test of all congeners indicated overall homogeneity

( >0.10 and 37 DOF). 3.2. Ecological Effects The ecological data representing the incidence of NHL versus PCB accumulation over time indicated statistically strong correlations ( 2>0.94) regardless of the assumed accumulation models (Figure 3 ). A high degree of correlation ( 2>0.8) between NHL incidence and PCB accumulation was observed for several other cancers for one or two of the accumulation models, including breast, liver and bile duct, kidney, skin, and soft tissue and heart, however none of these other cancers consistently demonstrated highcorre lation values in all three models (Table 5 ).

Table 5: Summary of the dose -response correlations between sy nchronized polychlorinated biphenyl (PCB) bioaccumulation and the incidence of cancer. Accumulation is represented as based on sales alone (sales growth), environmental exposure (static growth), and physiologic exposure (dy namic growth). Figure 3: Ecological correlations between PCB accumulation and the national incidence of NHL compared over calendar ye ars and relative ye ar based on the assumptions of (a)sales - only

exposure (sales model); (b) environmental exposure (static model); (c) physiologic exposure (dy namic model). 

### 4. Discussion The results of the (nonexhaustive) application of the Hill criteria to the current state of knowledge regarding the relationship between blood levels of PCBs and NHL risk indicate a plausible causal association. The multiple case - control studies described herein also consistently

demonstrate a strong level of association between elevated PCB levels and NHL sufficient to conclude that a (general) causal relationship exists between the two, and that if PCBs were to be e liminated from the environment, a certain proportion of NHL cases would likewise be eliminated. This conclusion is further supported by the results of the ecological analysis reported herein, in which a relationship between PCB levels in the environment over time appeared to be more uniquely and strongly correlated with NHL incidence than with other ty pes of cancers. The multicause nature of NHL means that these data should be viewed with caution and even skepticism, but the observed relationships are at the very least consistent with other theories regarding the inordinately l arge increase in NHL incidence over the past approximately 40 years. Based on these results and the multifactorial nature of NHL, it is reasonable to conclude that a certain proportion of individual cases of NHL occur only because of elevated PCB levels; in another proportion, elevated PCB levels have contributed to the cause of the NHL in conjunction with other causes but cannot be said to be solely necessary

as a cause; in another proportion the body burden of PCBs is neither com pletely nor partially contributory to the NHL occurrence. We posit that if an individual with NHL is found to have a titer of PCB congeners 118, 138, 153, 156, 170, 180 or 187 that exceeds 75% of that of the comparable general population of the same age and era [ 75 ], then it can be concluded that the elevated body burden of PCBs causally c ontributed to the NHL occurrence. Given this conclusion, the issue of the >2.0 relative risk specific causation threshold must be addressed. In a setting in which there is an identified unexposed comparison group, a relative risk of 1.43 (here defined by an OR with the lesser exposed) would mean that out of 143 hazard- exposed subjects with the disease of interest, 43 acquired the disease onl y because of the exposure, and 100 developed the disease independent of the exposure to the hazard. For the purposes of a specific causation evaluation, randomly s electing one of the exposed cases would result in a probability of causation of 30% (43/143), and thus the conclusion that the individual’s disease was not related to the exposure, on a more probable than not basis. Such an approach is potentially problematic b ecause it will result in an erroneous determination of no causal relationship between the hazardous exposure and the disease in 30% of specific causation evaluations [ 76 ]. The 2.0 relative risk approach becomes problematic to a point of impracticality when evaluating specificcausation for PCBs and NHL. When considering the 143 highest PCB titer percentile exposed subjects with NHL (representative of the meta- analyzed 1.43 ORMH described herein), there will be 43 who have the disease only because of the PCB exposure, and 100 subjects in whom their elevated PCB levels may or may not have contributed to their NHL, since in reality

all of the “unexposed” subjects are really j ust “l esser exposed” subjects. The fact that there are no unexposed comparison groups (zero PCB body burden) with which the cases could be compared in the meta- analyzed studies effectively lowered the resulting odds ratios to a largely unknown degree. Some indic ation of the magnitude of this effect can be inferred from the ecological data presented herein, as there was an approximate doubling of the incidence of NHL over a 30 - ye ar period of time that was temporally associated with the introduction of PCBs into the environment. It may be a reasonable supposition that some proportion of the cases resulted from the exponential increase in environmental PCBs that preceded the dramatic increase in the rate of NHL and which are represented in all percentiles of PCB expo sure. Additionally, it appears that there are a couple of factors that tend to decrease PCB levels in the body

that may be associated with the presence or diagnosis of NHL, resulting in lower post-NHL - diagnosis titers than what may have been presented prior to diagnosis. Individuals with higher body mass index (BMI) levels metabolize and eliminate PCBs more slowly, and because weight loss is a common feature of NHL, this feature of the disease would tend to decrease the body burden of PCBs as the illness progressed [ 77 ]. Additionally, chemotherapy, a common medical treatment for NHL, has been observed to potentially

decrease PCB levels in the body by nearly 30% [ 78 ]. Taken together, all of these factors indicate that the 2.0 relative risk or odds ratio threshold cannot be reasonably applied to PCB and NHL. It is for this reason that the use of upper percentile cutoff values associated with the maximum versus minimum ORMH to conclude that causal contribution is present is thought to be a reasonably practicable alternative to evaluating causation for an environmental toxin so ubiquitous that no unexposed group exists for comparison. 

### 5. Conclusions Application of the Hill criteria to the current state of knowledge regarding the association between environmental PCBs and NHL reveals convincing evidence of plausibility, as well as a strong general causal association, with meta- analyzed odds ratios indicating a 43% association of studied NHL cases in the literature with total PCB levels in the highest percentile, relative to comparison populations in the lowest percentiles of PCB levels. For evaluation of the causal contribution of PCBs to an individual case of NHL, the meta- analyzed values for seven immunotoxic congeners (118, 138, 153, 156, 170, 180, and 187) are presented and compared with relevant population survey data. When an individual case of NHL presents with one of these seven congener titers that fall into the highest quartile of their representative general population, it is reasonable to conclude a causal contributory

relationship is present, on a more probable than not basis.

### Disclosure

Both authors provide forensic consulting services for environmental toxin litigation. 

### Acknowledgments Partial support was provided by the National Institutes of Health (to SSK). The authors appreciate the valuable technical comments from Wendy M. Leith. 

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– 197, 2000. View at ScopusNumber of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1975 7.9 - 2.1 1976 8.2 - 2.2 1977 8.9 - 2.3 1978 8.9 - 2.3 1979 9.5 - 2.4 1980 10.5 - 2.3 1981 11.1 - 2.4 1982 11.2 - 2.5 1983 11.1 - 2.5 1984 11.4 - 2.5 1985 12.8 - 2.6 1986 13.3 - 2.6 1987 13.7 - 2.6 1988 12.9 - 2.6Number of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  1989 13.7 - 2.7 1990 13.9 - 2.8 1991 14.6 - 2.7 1992 14.8 14.1 2.7 1993 14.6 13.8 2.7 1994 15.7 14.8 2.7 1995 16.5 15.8 2.7 1996 17.4 16.5 2.8 1997 17.7 16.8 2.7 1998 17.9 16.8 2.8 1999 18.3 17.3 2.6 2000 19.0 17.7 2.7 2001 19.7 18.5 2.7 2002 19.3 17.9 2.6 2003 19.5 18.0 2.7Number of New Cases and Deaths Per 100,000 People (All Races, Males and Females), Age Adjusted **Year New Cases**  **- SEER 9**  **New Cases -**  **SEER 13 Deaths**  **- US**  2004 20.7 19.0 2.7 2005 22.5 20.4 2.8 2006 22.1 20.0 2.7 2007 21.8 20.1 2.7 2008 23.1 21.0 2.7 2009 23.0 20.5 2.8 2010 23.8 21.3 2.7 2011 22.7 20.3 -New Cases, Deaths and 5 - Year Relative Survival Vie w Data Table

**Year 1975**  **1980 1985**  **1990 1994**  **1998 2002**  **2006** 5 -Year Relative Survival 81.8% 83.9% 86.1% 89.2% 88.8% 90.9% 92.9% 92.8% SEER 9 Incidence 1975

- 2011 & U.S. Mortality 1975

- 2010, All Races, Both Sexes. Rates are Age

Adjusted.Béatrice Lauby - Secretan , Dana Loomis , Yann Grosse , Fatiha El Ghissassi , Véronique Bouvard , Lamia

Benbrahim -Tallaa , Neela Guha , Robert Baan , Heidi Mattock , Kurt Straif , on behalf of the International Agency for Research on Cancer Monograph Working Grou p IARC, Lyon, France In February 2013, 26 experts from 12 countries met at the International Agency for Research on Cancer (IARC), Lyon, France, to reassess the carcinogenicity of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). These assessments will be published as volume 107 of the IARC Monographs. 1 PCBs are a class of aromatic compounds comprisin g 209 congeners, each containing one to ten chlorine atoms attached to a biphenyl nucleus. Technical PCB products, which were manufactured to obtain a specific level of chlorination, are mixtures of many PCB congeners. These products were widely used as di electric fluid in capacitors and transformers, and to a lesser extent in building materials (eg, caulking, paints, and lighting ballasts). PCB production and new use were banned in most countries by the 1980s, but production has been reported recently in N orth Korea. Earlier, occupational exposure was highest during manufacture of PCBs, transformers, and capacitors; today, exposure can come from demolition, dysfunction, or uncontrolled recycling of PCB - contaminated structures and equipment. PCBs are persist ent and bioaccumulate; they have become ubiquitous environmental pollutants, including in polar regions and the deep ocean. Because of weathering and biotransformation, the PCB profiles noted in the environment or during biomonitoring differ from those of the commercial products. The general population is exposed mainly via food, mostly from contaminated animal fats; two major episodes of food poisoning took place in Japan and Taiwan, China, where cooking oil was accidentally contaminated with PCBs. Indoor air can also contribute to human exposure. Worldwide monitoring programmes have shown that PCBs are present in most samples of human milk. PCB congeners can be categorised by their degree of chlorination, substitution pattern, and binding affinity to recep tors. 12 congeners with a strong affinity for the aryl hydrocarbon receptor (AhR) are referred to as dioxin - like PCBs. PCBs are readily absorbed and distributed in the body, and accumulate in adipose tissue. Biotransformation of all PCB congeners starts wi th cytochrome P450 - dependent mono oxygenation. Low - chlorinated PCBs are readily metabolised into highly reactive electrophilic species (ie, arene oxides, quinones) which, in addition to producing DNA adducts and reactive oxygen species, are directly genoto xic and mutagenic. 2 By contrast, highly chlorinated PCBs are poorly metabolised but, through induction of xenobiotic - metabolising enzymes, can also generate reactive oxygen species, lipid peroxidation, oxidative and alkylating DNA adducts, and can eventually cause genotoxic effects.Individual PCBs activate numerous receptors, including AhR and the constitutive androstan e and

pregnane xenobiotic receptors (CAR/PXR). AhR activation is one of the key events linked to carcinogenesis mediated by dioxin - like PCBs. Sustained activation leads to deregulation of cell - cycle control and cell proliferation, inhibition of apoptosis, suppression of cell -to - cell communication and adhesion, and increased cell plasticity and invasiveness. Non - dioxin - like PCBs induce many of these effects via several AhR - independent mechanisms, including activation of the constitutive androstane and pregna ne xenobiotic receptors, and perturbations in cell -to - cell communication and cell adhesion. PCBs can compromise the immune surveillance mechanism. Highly chlorinated PCBs with a strong affinity for the AhR are potent immunotoxicants; less - chlorinated PCBs, which are less immunotoxic, act via AhR - independent mechanisms, including metabolic activation. Both low - chlorinated and high chlorinated PCBs are associated with chronic inflammatory responses. Non - dioxin - like PCBs can stimulate the production of inflamm atory mediators, whereas dioxin - like PCBs can inhibit such reaction. By contrast, some dioxin - like PCBs, but not non - dioxin - like PCBs, can compromise the normal function of the vascular endothelium. PCBs target the endocrine system. Several models have sho wn direct modulation of nuclear steroid hormone - dependent gene expression by PCBs. Furthermore, depending on their structure, monohydroxylated PCB metabolites can act as oestrogen agonists or antagonists. These disruptions might have reproductive, toxic, a nd carcinogenic consequences. The Working Group considered more than 70 independent epidemiological studies with informative data for carcinogenicity of PCBs in human beings. Excess risks for melanoma were reported in several studies, mainly cohort studies of workers in the manufacture of capacitors and transformers, and in electric power and equipment maintenance. A significant linear exposure — response trend was noted in the largest study. 3 In a population - based case - control study that assessed exposure with PCB serum levels, the association persisted after control for sun sensitivity and exposure. 4 The association of melanoma and PCBs was noted consistently in occupational studies in different industries in North America and Europe, i n studies of the general population, and with cohort and case - control designs. Thus, the Working Group concluded that there is sufficient evidence in humans for the carcinogenicity of PCBs. Notably, AhR can modulate melanogenesis, 5 which lends mechanistic plausibility to this association. Increased risks for non - Hodgkin lymphoma and breast cancer were also reported, both of which are biologically plausible. However, the associations were not consistent and were considered as providing limited evidence. Data for cancers at other sites were too sparse to draw any conclusions. The carcinogenicity of PCBs in animals has been assessed for individual congeners; binary mixtures of congeners; technical mixtures containing various congeners; and simulated environmental mixtures, with 2 year bioassays; studies with perinatal and postnatal exposure; and studies that examined the init iating and promoting activities of PCBs. Individual congeners (PCB118, PCB126) and several commercial products with a high chlorine content induced benign and malignant tumours of the liver, lung, and oral mucosa in rats; these studies provided sufficient evidence of carcinogenicity in experimental animals. 6 , 7 These congeners and mixtures include AhR agonists that exhibit dioxin - like activities, and CAR agonists. 8 Other PCB congeners (PCB153) and low - chlorinated commercial products,which were less well studied than highly chlorinated products, showed limited evidence of

carcinogenicity in experimental animals. 9 The relative contributions of different PCB congeners to the carcinogenicity of the commercial mixtures are not kno wn. Overall, all PCBs can induce formation of reactive oxygen species, genotoxic effects, immune suppression, an inflammatory response, and endocrine effects to various extents and through different pathways. The dioxin - like PCBs exert their effects mainly through AhR activation and the downstream cascade of related events; less - chlorinated PCBs act more readily through metabolic activation and the downstream effects of these metabolites. Thus, mixtures might have more than additive effects. On the basis of sufficient evidence of carcinogenicity in humans and experimental animals, the Working Group classified PCBs as carcinogenic to humans (Group 1). Additionally, dioxin - like PCBs were also classified in Group 1 on the basis of extensive evidence of an AhR - m ediated mechanism of carcinogenesis that is identical to that of 2,3,7,8 -tetrachlorodibenzo - para - dioxin, and sufficient evidence of carcinogenicity in experimental animals. However, the carcinogenicity of PCBs cannot be solely attributed to the carcinogeni city of the dioxin - like PCBs. PBBs resemble PCBs, with bromine rather than chlorine atoms. PBBs were used mainly as flame retardants in the 1970s, but production has been discontinued in most countries for many years. One release of PBBs took place in Mich igan, USA, where they were inadvertently distributed as cattle -feed supplement, contaminating many agricultural products. Similar to PCBs, PBBs are highly lipophilic, bioconcentrate and bioaccumulate, and are environmental contaminants worldwide. FireMaste r FF - 1, the most widely used commercial PBB product, consistently induced benign and malignant hepatocellular tumours in rats and mice, and cholangiocarcinomas in rats. 10 PBBs, like their chlorinated analogues, are ligands to several cellular and nuclear receptors, including AhR. They are efficacious inducers of hepatic drug metabolism, accelerating the biotransformatio n of both endogenous and exogenous compounds. PBBs have various adverse effects including suppression of the immune system and disruption of normal hormone function. They are efficient promoters in two - stage rodent hepatocarcinogenesis bioassays. Although PBBs have received less attention and study, the available data indicate that PBB congeners exhibit their toxic effects and carcinogenic potential via many of the same pathways as their chlorinated counterparts. On the basis of these similarities with PCBs , and together with inadequate evidence for carcinogenicity in humans and sufficient evidence in experimental animals, PBBs were upgraded to Group 2A, probably carcinogenic to humans References 1 IARC. IARC monographs on the evaluation of carcinogenic risks to humans, volume 107. Polychlorinated and polybrominated biphenyls. Lyon: International Agency for Research on Canc er (in press). 2 Ludewig G, Robertson LW. Polychlorinated biphenyls (PCBs) as initiating agents in hepatocellular carc inoma. Cancer Lett 201210.1016/j.canlet.2012.11.041. published online Dec 2. PubMed3 Loomis D, Browning SR, Schenck AP, et al. Cancer mortality among electric utility workers exposed to

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