AMERICANJOURNAL

### OF INDUSTRIAL MEDICINE29:236-246 (1996)



# HematotoxocityAmong Chinese Workers Heavily Exposedto Benzene

Nathaniel Rothman, MD, MPH, MHS,

### Gui-Lin Li, MD, 

### Mustafa Dosemeci, PhD, 

### William E. 

### Bechtold, PhD, 

### Gerald E. Marti, MD, PhD, 

### Yao-ZuWang, MD, 

### Martha Linet, MD, 

### Li-qiang

### Xi, MD, 

### Wei Lu, MD, 

### MartynT. Smith, PhD, 

### NinaTitenko-Holland, PhD, 

### Luo-Ping Zhang, PhD, 

### William Blot, PhD, 

### Song-NianYin, MD, 

### and Richard B. Hayes, PhD



### KEY WORDS: 

### benzene, hematotoxicity, phenol, rnuconicacid, 

### Itydroquinone, calechol

Abbreviations: GUMS, gas chromatography/massspectroscopy: WBC. white blood cell; MCV, mean corpuscularvolume; TWA, time-weighted average; ppm, parts per million: SHAC, Shanghai Hygiene and Anti-Epidemic Institute; NCI. National Cancer Institute; RBC, red blood cell.

Epidemiologyand Biostatistics Program, National Cancer Institute, Bethesda, MD (NR.. M.D.. M.L.. W.BI.. R.B.H.).

Chinese Academy of Preventive Medicine, Instituteof Occupational Medicine, Beijing, China (G.-L.L.. L.-Q.X.,S.Y.).

Lovelace InhalationToxicology Research Institute.Albuquerque, NM (W.Be.). Center for Biologics Research and Evaluation, Federal Drug Administration, Bethesda, MD (G.E.M.). Shanghai Hygiene and Anti-Epidemic Center, Department of Occupational Universityof California,School of Public Health, Berkeley, CA (M.T.S, N.T.-H.,
Address reprint requests to Dr. Nathaniel Rothman, NlHlNCl EPN 418, BeAccepted for publication March 17, 1995. Health, Shanghai, China (Y.-Z.W.. W.L.). L.-P.Z.). thesda, MD 20892.



### (C)

1996Wiley-Liss, Inc. 'This article isa US Governmentworkand,as such, is inthe public domain inthe United States of America.Hematoxicity and Benzene 

### 237



### INTRODUCTION

Benzene has been known as a hematotoxin for more than a century [reviewed

### in Aksoy. 1988:Goldstein. 19771. Although benzene has been shown to cause pancytopenia and aplastic anemia. the relative sensitivity of specific peripheral blood cells to benzene. in the absence of severe marrow toxicity, is uncertain. Discrepancies 

### in the older literature may have been due to variation in the method of measuring blood cells, exposure to additional compounds such as toluene [Andrews et al., 19771 which may affect benzene metabolism, 

### or failure to adjust for potential confounders. As part of a large cohort study of cancer and hematotoxicity among approximately 75,000 workers exposed to benzene sponsored by The Chinese Academy 

### of Preventive Medicine and the U.S.National Cancer Institute [NCI;Yin et al.. 19951, we performed a cross-sectional study of currently exposed workers 

### in order to gain insight into early biologic effects associated with benzene exposure. Here,we report the association between benzene exposure. benzene metabolite formation, and hematotoxicity. 

### MATERIALSAND METHODS Factory Identificationand Subject Enrollment Workplaces were identified from public health district records and from exposure and demographic data previously collected for the cohort study 

### [Yin et al.. 19951. Exposed factories were selected so that the study population would have a wide range 

### of exposuresto benzene (I to 2 25 parts per million [ppm] as an 8-hr time-weighted average [TWA],similar to that found among workers 

### in the cohort study)? minimal exposure totoluene. and no other exposures to known hematotoxic 

### or genotoxic agents. Factory I used benzene to solubilize natural rubber for subsequent production of rubber padding for printing presses. Factory 2 used benzene to manufacture adhesive tape. In Factory 3, benzene-based paint and varnish were applied to wooden toys and boxes. Two workplaces 

### in the same geographic area that did not use benzene or other chemicalsassociated with bone marrow toxicity were selected as control factories. One factory manufactured sewing machines while the second workplace was an administrative facility. Study subjects were enrolled in the fall 

### of 1992 

### in Shanghai, China. Exclusion criteria for all study subjects included prior history of cancer,therapeutic radiation. chemotherapy. 

### or current pregnancy. Eligibility criteria for exposed subjects were current employment for at least 6 months in a factory which used benzene, minimal exposure to other aromatic solvents, and 

### no exposureto other known marrow-toxic chemicals or ionizing radiation. Eligibility criteria for controls were 

### no history of occupational exposure 

### to benzene. other marrow-toxicchemicals. or ionizing radiation. Unexposed controls were frequency-matched to the exposed subjects on age (5-year intervals) and gender.



### Industrial Hygiene Assessment
Current exposure assessment

Individual exposure was monitored by organic vapor passive dosimetry badges 

### (3M no. 3500. St. Paul. Minnesota). which were worn by each worker for a full workshift
on 5 separate days during the I-?-week period prior to phlebotomy. Badges were analyzed at the Shanghai Hygiene and Anti-Epidemic Institute (SHAC)by gas chromatography (GC)with flame ionization detection forbenzene, toluene. and xylene. An 8-hrTWA exposure was calculated for each compound as the geometric mean of the five air measurements. Only trace amounts of other aromatic solvents were detected in analyses. As a quality control check. two badges were worn for a 

### full workshift by each of I0 workers who were exposed to a wide range of benzene levels. One badge was analyzed by the SHAC and the duplicate badge was analyzed by ClaytonEnvironmental Consultants (Novi. Michigan).The I0 duplicate measurements were essentially identical (Spearman r = 0.98. p 

### <0.0001). A detailed assessment of factory records showed that
no other known marrow-toxic chemicals or physical agents
were present 

### in these workplaces. Sixteen workers 

### in Factory 1 used half-mask charcoal-activated respirators during the highest exposure period 

### of their workshift, which lasted about 3 hr per shift. Neither quantitative nor qualitative fit testing was performed. Workers typically changedthe charcoal once every 

### 1-2 days or more frequently. depending upon when they smelled benzene inside the respirator. 

### In a preliminary analysis. factory type and respirator use did not alter the association between benzene exposure and hematologic outcomes. Therefore, neither of these variables was included 

### in the subsequent analyses. Forty-three of 44 benzene-exposed workers provided a spot urine sample at the end of a workshift (Factories

### 2, 3)


### or at the end of the high exposure period during their workshift (Factory I). Samples were immediately aliquoted and frozen on dry ice. and transported back to the SHAC 

### for storage and later shipment to an NCI biorepository 

### in the United States. Themajority of subjects 

### inthe control factory that manufactured sewing machines were monitored for air levels of benzene. toluene, and xylene for I day on any of 6 different samplingdays. 

### No monitoring was performed 

### in the second control workplace which was an administrative facility. Control subjects provided a spot urine sample at the SHAC during the clinical phase of the study.238 Rothman 

### et 

### al.



### Historical exposure assessment
Historical benzene exposure during subjects' employment at the study factories was estimated by trained field personnel using methods similar to those developed to 

### assesshistorical exposure

### inthe cohort study [Dosemeciet 

### al., 19941. This estimate incorporated work histories obtained by interview and review of workplace employment records, benzene area measurements obtained from factory records, and information 

### on annual amount 

### of benzene used, percent benzene used 

### in workplace solvents, changes 

### in manufacturing, and ventilation practices. Historical estimates considered current individual exposure levels when historical exposure monitoring data were sparse. Estimates of cumulative exposure 

### to benzene were calculated from the historical time-specific exposure estimates and 

### the duration worked. Exposure assessment was performed blinded with respect to hematologic measurements. 

### Interview and Biologic Sample Co

### Ilect

## ion
Workers were evaluated at the SHAC. The study protocol was explained to all potential participants, and 

### informed consent was obtained using Institutional Review Board-approved procedures. Eight-eight 

### of 94 (94%)cligible subjects selected for study agreed to participate. Each subject was administered 

### a questionnaire by a trained interviewer from the staff of the Department of Occupational Health. Data collected included age, gender, current, and lifelong tobacco use, alcohol consumption,medical history, and an occupational work history. Height and weight of each subject were measured and a 27 mL sample of blood
was obtained by venous phlebotomy. Peripheral blood films were made immediately after phlebotomy with blood that had not been anticoagulated. A spot urine sample was collected and analyzed by gas chromatography/mass spectroscopy (GC/MS) 

### for cotinine levels and adjusted for creatinine; these were highly correlated with self-reported current number of cigarettes used per day (Spearman r = 0.86,p 

### < 0.000l). 

### Blood Cell Analysis

Blood samples were delivered within 4-6 hr after collection 

### to the Huang Shan Hospital in Shanghai.Blood 

### sanples were analyzed by a Coulter TS40blood counter which
was calibrated daily. Approximately 10' white blood cells (WBC; Coulter Corporation, personal communication) are counted to provide a total WBC count and a lymphocyte percentage, which are multiplied together to generate the absolute lymphocyte count. All abnormal counts were reviewed by hand. Five hematologic measurements are presented 

### in this paper; the total WBC count (xIO-'/pL blood), the absolute lymphocyte count (x103/pL blood),the hematocrit (%). the red blood cell count (RBC;xlOh/pLblood), and the platelet count (x103/pLblood). The mean corpuscularvolume (MCV;pm') was calculatedasthe hematocriv (RBCcount x 10).TheWBC differential was hand-counted


### on 100 cells by technician5 at the American Medical Laboratories, Inc. (Chantilly, Virginia). Peripheral films were systematically reviewed by a hematologist (G.E.M.) and scored using a standardized detailed form for RBC, WBC, and platelet morphology. Since there were a wbstantial number of subjects with automated platelet counts below 100x IO'/pL blood 

### inboth the exposed and control groups, the average number of platelets per high powered field was counted as a quillity control check; there was a positive correlation (Spearman r = 0.65, p = 0.001)between these two measure$. 

### Benzene Metabolite Measurements

Benzene urinary metabolites were measured (W.E.B.) using a modification of a previously described isotope dilution GUMS assay [Bechtold et al., 19911. "C-labeled analogues of the trans,trans-muconic acid (niuconic acid), and phenol. catechol, and hydroquinone as sulfate and glucuronide conjugates, were used 

### as internal standards. Concentrations were determined by calibrating against a primary standard solution. Urine samples were diluted with deionized water because of the high concentrations of metabolites. Thus, 200 

### pL of distilled water were added to vials with 

### 200 pLof urine samples, followed by 100pL of internal standard solution. The contents were mixed with 100 pL 

### of concentrated HCI. The vials were capped and incubated at 

### 100°C for 

### 60 min, and allowed 

### to cool. The solution was extractedwith 

### 2 niL of ethyl acetate.Theethyl acetate layer was removed and dried with a small scoop of sodium sulfate. The sample was decanted and the drying agent was rinsed with 1 mL of ethyl acetate. The ethyl acetate aliquots were combined and evaporated to 200 

### pL under nitrogen. The analytes were derivatized with 100pL of 

### bistriniethylsilytrifluoracetamide, incubated at 60°C for 30 min. and analyzed by GC/MS. 

### Statistical Methods

Arithmetic mean L-standard deviation (ad) and median with range areused to suniniarize data.Correlation analyses were performed with Spearman rank order correlation.The Wilcoxon rank sum test was used to test for differences in hematologic outcomes between exposed subjects and controls. Subsequent analyses were performed 

### on three groups 

### of study subjects: controls, subjects exposed to 531 ppm benzene 

### (the median TWA exposure level), and subjects exposed to 

### >31 ppm benzene. The distribution 

### of selected hematologic outcomes within these exposure categories is shown with Box and Whisker plots [Tukey, 19771.Contin-Hematoxicity and Benzene 239

TABLE I.

### Demographic 

### Characteristics of 

### Workers Exposed 

### to 

### Benzene and 

### Controls, Shanghai, 

### China, 1992
Exposed Controls 

### (n= 44) In = 44) Mean f sd Mean ? sd
Age (years) 35.3

# *7.8 35.4 f7.3

Females (n=21) 0.0 0.0 Cigarettes/day

Males (n=23) 11.Of7.8 13.5 f13.7 Drinks of alcohollweek 1.5 k3.2 1.4 f2.2 Quetelet's index (weight,,/height,,,,,,2) 21.6 f3.2 21.5 f2.3

uous outcome data that were not normally distributed were logarithmically transformed to improve normality. Analysis of covariancewas used to test for group differences 

### in continuous outcomes. The original matching variables, age and gender, were included 

### in all models. Other variables tested for potential confounding 

### or effect modification included current smoking status (average number of cigarettes per day 

### in last month), current alcohol intake (categorized as 

### 0
or >I drink per week), and Quetelet's index. a measure of obesity calculated as weight,,lheight,,,,,,,'. Unconditional logistic regression was used to test for differences 

### in the proportion of subjects with a peripheral blood cell morphologic abnormality among exposed workers vs. controls, and to evaluate potential confounders. Two-sidedp values were calculated throughout:p values 

### < 0.05were considered adequate 

### to reject the null hypothesis. All statistical analyses were performed using SAS for Personal Computer version 6.04 [SAS Institute Inc., 19871. 

### RESULTS 

### Subject Characteristics
Forty-four workers currently exposed to benzene and 44 never-exposed controls were enrolled 

### in the study. Age, alcohol intake, Quetelet's index, and daily cigarette use were comparablebetween exposed and control subjects (Table I).Twenty-oneof 44 (48%) subjects

### ineach group were female. 

### ExposureAssessment

Mean (sd) years of occupational exposure to benzene among the exposed subjects were 6.3 (4.4),with 

### a range of 0.7-16 years. Benzene comprised 30-44% of the primary solvent mixture used 

### in !he exposed factories. The median 8-hrTWA benzene personal air level was 

### 31 ppm. Exposure to toluene and xylene was low, relative to benzene (Table 11). As shown 

### in Table 111, the amount of each benzene metabolite 

### in urine was substantially higher among exposed than control workers.Among the 38 exposed subjectsmonitored for both air exposure and urine metabolite levels on the same day, there was a significant correlation (p 

### < 0.0001)between benzene 

### air levels and phenol, muconic acid, hydroquinone,and catechol urine levels(r=0.71,0.66 0.68, and 0.66. respectively). Similar correlations were observed in men and women (e.g., for phenol: 

### r = 0.69, p = 0.002 among 17 men and 

### r = 0.61, p = 0.003 among 

### 21 women). 

### Also noteworthy, the relative increase 

### in metabolite level 

### in the group exposed at greater than the median benzene exposurevalue (3I ppm) comparedto thoseexposedto less than this amount was about 9-fold for phenol and catecho1 and only 5 to 6-fold for hydroquinone and muconic acid (Table 111). The proportion of all measured benzene metabolites excreted as muconic acid and hydroquinone decreased from 

### a median (range) of 32% (21-42%) 

### in workers exposed to 531 ppm benzene to 24% (14-39%) 

### in workers exposed to >31 ppm (p= 0.006 by Wilcoxon rank sun1 test). 

### Hematologic Measurements
Automated peripheral blood cell counts

The absolute lymphocyte count was the most significantly decreased blood cell measure 

### in the exposed group compared to controls (mean [sd] I

### .5 [0.3]x 

### IO'/pL vs. 

### I .9 [0.4]x 

### 103/pL, p 

### <0.0001).Other measures also decreased
were the platelet count (127 [44] x 

### IO'/pL vs. 166 [59]x 

### IO'/pL, p =O.OOl), WBC count (6.0 [ 1.91x 

### I03/pLvs. 6.8 [ 1.71x 

### IO'/pL, p = 0.006),RBC count (4.4 [0.6] x 

### I06/pL
vs. 4.7 [0.6] 

### x 106/pL,p = O.Ol), and hematocrit (40.0% [5.6] vs. 42.0% [5.6], p 

### = O.lO), while the MCV was significantly elevated (91.4 [4.01 

### pm' in exposed subjects compared to 88.9 (4.91

### pm3in controls, p = 0.02). These differences were minimally changed after adjustment for alcohol consumption, cigarette use, and Quetelet's index. Comparable results were observed 

### in men and women. 

### For example, the absolute lymphocyte count was 

### a more sensitive measure 

### of benzene exposure than the 

### total WBC count 

### in both groups. The total WBC count 

### (xIO'/pL) was 9% lower

### in exposed women vs. female controls (mean [sd] 6.1 12.01 vs. 6.7 [2.2]. p = 0.16). while the absolute lymphocyte count 

### (x103/pL) was 22% lower

### inexposedwomen compared to female controls 

### (1.4[0.4] vs. 1.8 [0.3], p = 0.002). Similarly, the total WBC count was 12% lower in exposed men 

### vs. male controls (6.0 [ 1.81vs. 6.8 [1.21,p = 

### O.OI), while the absolute lymphocyte count was 25% lower 

### in exposed men compared to male controls 

### (1.5 [0.32]vs. 

### 2.0 [0.4], p = 

### 0.0005). Table IV portrays summary values for blood counts 

### in240 Rothman 

### et al.

TABLE 

### 11. 

### Current

### Benzene. 

### Toluene, and 

### Xylene 8-HrTWA 

### Personal Air Levels (pprn),by 

### Exposure Category, 

### in 

### a Study of 

### Workers Exposed 

### to 

### Benzene in Shanghai, China, 1992

Benzene(ppm) Toluene (ppm) (PPm) Median (range) Median (range) Median (range)
Control (n=44)a 0 02 (001-0 1) 002 (002-0 3) 0 02 (002-0 6) Exposed

5 31 ppm 

### (n = 22)’
> 31 ppm (n = 22) 136 (16-30 6) 91 9 (31 5-328 5) 0 02 (001-9 7) 002 (002-1 2 8) 0 2 (003-7 2) 0 1 (002-12 9)

lAir samples obtained for 31 of 44 controls (one sample) ’Air samples for exposed sublects represent geometric mean 01 live samples

TABLE 

### 111. 

### Benzene Urine Metabolites,by 

### ExposureCategory, in 

### a Study of 

### Workers Exposed to 

### Benzene in Shanghai, 

### China, 1992’

Phenol Calechol Muconic acid Hydroquinone Median (range) Median (range) Median (range) Median (range)

~~ ~ ~ ~ Control (n-44)a 17 3 (37-55 3) 3 2 (16-8 7) 0 18 (0 1-0 8) 1 6 (09-6 7) Exposed

531 ppm (n = 22)b >31 pprn (n = 22)c 38 9 (15 2-217 1) 349 7 (368-516 7) 7 0 (25-38 9) 65 7 (5 0-84 2) 8 15 (2 3-55 5) 46 8 (86-103 4) 128 (32-61) 64 3 (97-196 6)

‘Units pg/mg creatinine for all metabolites “A subsarnple 01 17controls frequency matched on age and gender to the exposed workers were tested lor urine metabolites in urifie collected while at the SHAC clinic ’Twenty two/22 sublects provideda urine sample ‘Twenty-one/ZZ subjects provided a urine sample

unexposed controls and among subjects exposed to less than and greater than the median benzene level (31 pprn). The respective absolute lymphocyte count and MCV distributions are portrayed in Figures 1 and 

### 2.All blood parameters
were significantly different between controls and sub.jects cxposed to >3 I pprm (Table IV).The magnitude of the diflerence was largest for the absolute lymphocytecount 

### (32% decrease)and smallest for the MCV (4%. increase).Only the absolute lymphocyte count. 

### RBC count, and platelet count
were significantly different between controls and subjects exposed to13I pptn benzene. These group differenceswere niininially changed after adjustment for alcohol consumplion, cigarctte use, and Quelelet‘sindex. Sirice the lower exposed group (131 ppm) contained
some subjects with 8-hr TWA exposures above 3I ppni on at least one of five sampling days. a subgroup was created that contained sub,jects who never had a sample above 

### 31 ppm on any sampling day 

### (n= I I,median 8-hr TWA =7.6 ppm. range 

### = 1-20 ppm). Only the absolute lymphocyte count differed significantly between this subgroup and controls (mean Isd] 1.6 [0.4]vs. 1.9 10.41.p = 0.03). In an analysis restricted tothe benzene-exposed workers, we examined the strength ofthe exposure-response relationship overthe entire range 

### ofexposures as indicated by individual air and urinary metabolite nieiisiirements (Table V). The urine nietabolite data compliment exposure asscssed hy the 8-hr TWA benzene air measurements since they incorporate interindividual variation 

### in respirator use, percutaneous absorption ofbenzene, and metabolism.Only the total WBCcount, absolute lymphocyte count,and MCV showed evidencefor a dose-response relationship with these
measures of benzene exposure. Similar results were generally found using the 8-hr TWA and urine metabolite data (Table V). Although the correlation between each benzene metabolite and the absolute lymphocyte count was similar (TableV),only the association with hydroquinone achieved statistical significance. 

### In contrast. catechol was inore highly correlated with the MCV 

### (r= 33.p 

### = 0.0001)than the other three benzene metabolites (range: 

### r =0.30 to 

### r = 0.36)(Table V)for the entire 

### group. Various combinations of urine metabolites had no stronger association with the hematologic outcomes than did levels of individual metabolites. Neither estimated cumulative life-time benzene exposure nor number of years worked in an exposed factory was significantly associated with any hematologic outcome (data not shown).Hematoxicityand Benzene 241

TABLE IV. Comparison of Peripheral Blood Counts, By Exposure Status, in a Study of Workers Exposed to Benzene in Shanghai, China, 1992'

Absolute lymphocyte WBC count RBC Hematocrit MCV Platelets Mean ksd Mean fsd Mean fsd Mean fsd lean? sd Mean tsd

(range) (range) (range) (range) (range) (range)
Control (n = 44) 6.8 ir 1.7 1.9t0.4 4.7 f0.6 42.0 t5.6 88.9 f4.9 166k59
Exposed (3.1-12.5) (1.1-2.8) (3.0-5.6) (27.7-50.7) (71.2-96.6) (62-313)

531 pprn 6.4 f1.8 1.6f0.3 4.6k0.6 41.2 t5.7 89.8 f3.9 132f45 (n = 22) (3.5-11.1) (1.1-2.5)a (3.2-5.7)b (29.5-49.7) (80.1-99.8) (59-204)b
>31 pprn 5.6 f1.9 1.3 t0.3 4.2 f0.6 38.8 f5.3 92.9 f3.4 121f43 (n = 22) (3.8-1 1.4)a (0.9-2.3)' (3.5-5.3)' (31.6-50.9)b (85.7-98.3)' (65-216)a

'Units: WBC count (x103/pL blood), absolute lymphocyte count (x103/pL blood), RBC count (x106/pL blood), hematocrit (YO). platelet count (x103/pL blood), MCV (prn3) ap < 0.01 bp < 0.05.

co < 0.001.


### Peripheral blood film differential and morphology

Theproportion of granulocytes in the WBCdifferential count increased from 

### 64% to 71% between controls and subjects 

### in the higher exposure group, lymphocytes decreased from 27% to 23%. and basophils decreased from 0.58to 0.1%.The percent monocytesand eosinophils were similar between these two groups, and no left shift among the granulocytes was observed (data not shown). A direct comparison of granulocyte vs. lymphocyte toxicity was made using data from the manual differentials by comparing the percent granulocytestimes the total WBCcount with the percent lymphocytes times the total WBC count. The absolute granulocyte count 

### (x103/pL)decreased only 7%, from
mean (sd) 4.3 ( 

### 1.7)among workers 

### in the 13 I ppm groupto 4.0 (1.8)among workers in the higher exposure group (p= 0.28). whereas the absolute lymphocyte count (x1

### 03/pL) decreased 25%. from 

### I .6 (0.6)to 

### 1.2(0.6) (p = 0.02). There were no significant differences observed overall
or for specific types of morphologic abnormalities 

### in lymphocytes, granulocytes, or platelets between controls and either of the two exposure groups (Table VI). 

### In contrast, there was a significant difference 

### in the proportion of subjects among the higher exposed group with any RBC abnormality compared to controls (86% vs. 5096, p 

### < 0.01). Significant elevations were seen specifically for stomatocytes and target cells 

### in the more highly exposed workers (Table VI). Thesedifferences were minimally changed after adjustment for alcohol consumption, cigarette use, and Quetelet's index. 

### DISCUSSION
Forty-four workers exposed to 

### a wide range ofbenzene were found to have decreased levels of all blood elements tested. as compared to controls. 

### The absolute lymphocyte count was the hematologic measure most significantly different between exposed and control subjects. and was the only blood cell type significantly lower 

### in a subgroup of workers with median TWA = 7.6ppm (range 1-20 ppm) compared to controls. Other hematologic parameters were altered 

### in the exposed group compared to controls and are consistentwith hematotoxic effectsof benzene reviewed by Aksoy [19881and Goldstein [ 19771,i.e.,toxicity for WBC, platelets. RBC, and a tendency towardsmacrocytosis. However. 

### in our study, none of these measures was as sensitive
an indicator of benzene exposure as the absolute lymphocyte count, particularly at levels 131ppm. Among the benzene-exposed workers, only the WBC count, absolute lymphocyte count, and MCV were significantly correlated with continuous (vs. categorized) measuresof benzene exposure.Thefinding of overall decreases among benzene-exposed workers of other peripheral blood cells, and yet the lack of a dose-responserelationship, may reflect 

### a more idiosyncratic reaction to benzene exposure.In addition, 

### in morphologic studies. we found that only RBC abnormalities, and not those ofany type 

### ofWBCorplatelet, were associated with benzene exposure,and only amongthe most heavily exposed workers. Goldwater [194I] noted that lymphocytopenia was
more frequently found among subjects exposed to benzene than was leukopenia. Aksoy found that 

### 28 of 37 leukopenic242 Rothman et

### al.

I 2.8 -

2.6

# I

2.4 -

Absolute Lymphocyte Count
( 10 '/pL blood)

1.8 -

16

1.4 0

0

-+-



# I

0.8 - 

### I I /I 0.0 - Exposure group: Control 5 31 ppm 31 ppm (n) (44) (22) (22)

FIGURE 1. Absolute lymphocyte count, by current benzene exposure category, inastudy ofworkers exposedto benzene in Shanghai, China, 1992. Upper edge of box represents highest 25th percentile of data. Lower edge of box represents 75th percentile. Upper and lower lines

patients exposed to benzene also had lyniphocytopenia [as described in Aksoy, 19881, though he did not address the relative sensitivities of these two measuresfor benzene toxicity. Yin et ill. [

### lY871 reported that the absolute lymphocyte count and leukocyte count were similarly reduced in workers exposed only to benzene. In contrast. several authors have reported either 

### no association between benzene and the absolute lymphocyte count. 

### or lymphocytosis [Bernard. 1942; Hamilton-Patterson and Browning, 

### 1934;Hernberg et al., 1966; Doskin, 1971; Aksoy et al., 19711. Although our study is relatively small compared to some of these previous reports, its strengths include comparison to matched controls, strict blinding in the analysis, (whiskers)indicate rangeof remaining dataexceptfor outliers. 0,outlier defined as >1.5 and ~3.0 x distance between 25th and 75th percentiles.

## +, mean; -----,median.

detailed recent and historical exposure data, a population exposed to benzene with relatively low levels of toluene exposure, and absolute lymphocyte counts measured by 311 automated Coulter counter. Further, we demonstrated a dose-response relationship between the absolute lyniphocyte count and benzene air level and benzene urine metabolites. and evaluated each association for potential confounding. Our findings are consistent with resultsof animal studies demonstrating sensitivity 

### of lymphatic tissue to henzene [Snyder et al., 1980;Ward et al., 19851at concentrations as low as I0 ppm benmie [Buarson et al., 19831.with numerous reports that have described an excess ofchrornosome damage inlymphocytes 

### from workers exposedto ben-Hematoxicity and Benzene 

### 243

loo

# i

98 -

96

# I

94 - 3 MCV 

### (pm /red cell )92 -

90

88

86

84 - 

# I

82 - 

# i

78 - 

### I It



### I

72

# i

70 - I

.-+..



## -

0



### * 

### * 0



# lr 

# 1

~ .+.. 

# T

0 

# 1

~ I I I Exposure group Control 5 31 ppm > 31 pprn (n) (44) (22) (22)
FIGURE2. Red blood cell mean corpuscularvolume, by currentbenzene exposure category,

### in a study of workersexposedto benzene

### in Shanghai,China, 1992.Symbolsas

### for Fig. 1., plus *extreme outlierdefined
as >3.0x distance between 25th and 75th percentiles.

zene 1e.g..Forni et al., 1971a,b; Picciano, 1978;Ding et al., 1983: Sarto et al., 19841, and with findings from several studies which suggest that benzene is associated with excess risk of hematopoietic tumors derived from lymphocytes [Rinsky et al.. 1987: Wong. 1987;La Vecchia et 

### al., 1989; Blair et al.. 1993; Yin et al., 19951. Most recent hematologic studies ofworkers exposed to benzene and unexposed controlshave not reported data 

### on the absolute lymphocyte count,including studies 

### of workers exposed up to 1.4ppm [Collinset al.. 19911. 10ppm [Yardley-Jones et 

### al.. 19881. 12.4ppm [Sartoet al.. 19841, 

### 31.5 ppm [Hancock et al., 19841, and 

### 35 ppm benzene [Townsendet al., 19781.Theseauthorsgenerally concluded that there is little evidence of benzene-induced hematotoxicity at these exposure levels, with the possible exception 

### of mild macrocytosis [Collins et al., 1991; Yardley-Jones et al., 19881or small declines in the RBC count [Townsendet 

### al.. 19781.Given the limited published data on the absolute lymphocyte count 

### in workers exposed to benzene in the 1-30 ppm range, particularly in the presence 

### of other aromatic compounds such 

### as toluene. it is difficult to evaluate the sensitivity of this measure at these exposure levels.244 Rothrnan et al.

TABLE V. Correlation of Peripheral 

### Blood 

### CountsWith Various Measures of Benzene Exposure 

### Among 

### Exposed Workers, Shanghai, 

### China, 1992'

Absolute Measures 01 WBC lymphocyte RBC Hematocrit MCV Platelets benzene exposure (R) count (R) (R) (R) (R) (R)
Air measurement
Urinary metabolites

8-Hr TWA -0 35a -0 3Ea -0 06 -0 01 033a -013

Phenol (n=43) -0 23 -0 31 -0 09 000 030 -002 Muconic (n=43) -0 23 -0 30 -0 11 0 00 0 3Za -0 0006 Hydroquinone (n = 43) -0 26 -0 36a -0084 0 00 036a 0016 Catechol (n = 42) -0 28 -0 25 -0 14 000 0 53b -0 048

'Correlation calculated by Spearman rank order correlation for all outcomes except hematocrit and RBC count, for these correlation coefficients adjusted for age and sex were determined froma linear regression model 'p < 005 bp < 0001

TABLE VI. Peripheral Blood Cell Morphology,by Exposure Category, 

### in 

### a Study of Workers Exposed

### to Benzene 

### in Shanghai, China, 1992
Granulocyte Lymphocyte abnormality abnormality No. No. abnormal/ abnormall

total total
Control (n = 42) 3/42 (7%) 7/40 (17%) Exposed

531 ppm (n = 22) 1/20(5%) 2/21 (10%) Platelet abnormality No. abnormall

total
9/40 (22%)

5/19 (26%) ABC abnormality Stomatocytes Target cells Anisocytosis No. NO. No. 

### No. abnormal/ abnormal/ abnormal/ abnormal/

total total total total
21/42 (50%) 11/42 (26%) 6/42 (14%) 22/42 (52%)

8/22 (36%) 6/22 (27%) 4/22 (18%) 15/22 (68%)
>31 ppm (n = 22) 0/21 (0%) 1/21(5%) 5/21 (24%) 18/21 (86Y0)~ 12/21 (57'/0)~ 8/21 (38y0)~ 16/21 (76%)

ap < 0.01. "p < 0 05.

In our study. the proportion of measured benzene metabolites excreted as muconic acid and hydroquinone decreased from 32% in workers exposed to 23I ppm benzene to 23% in workers exposed to 

### >31 ppm. The tendency to form less muconic acid and hydroquinone relative to phenol itnd catechol at higher benzene levels has also been nbserved in several animal studies [Henderson el 

### al.. 1989; Sabourin et al., 19891.The risk-assessment implications of shifts in benzene metabolite formation are uncertain, however, since the relativc contribution that each metabolite makes to the genotoxic effectsof benzene is still a subject of some debatc. While several studies have shown that benzene metabolites related 

### to muconic acid (e.g..trans, transmuconaldehyde) and hydroquinone and its related conipounds 

### (e.g.. p-benz.oquinone) are more genotoxic than phenol and catechol in some tests systems [Irons and Neptun, 1980; Rushmore el al., 1984; Schwartz et 

### al., 1985: Eastmond et al., 

### 1987; Snyder et al.. 1989; Glatt et al., 19891,catechol has been shownto he ii more potent inducer of sisterchromatid exchangethan hydroquinone [Erexson et al., 1985; Glatt et al., 1989;Morimotoet al., 19931.Further, phenol [Subrahmanyam et al., 19901 has been shown to stimulatehydroquinone bioactivation and potentiate hydroquinone-induced myelotoxicity (Eastinondet al.. 19871,and catechol has been shown 

### to stimulate hydroquinone bioactivation [Subrahmanyamet 

### al., 19911and potentiatehydroquinone-induced micronuclei formation [Robertson et al., 1991J. Thus, the complex nature 

### of benzene metabolite interactionsprecludes tirm conclusionsabout the relevance of alterations

### inbenzene metabolite formation at different levels 

### of benzene exposure. Since the four benzene urine metabolites measured 

### in this study were highly correlated (at about 90%),we had limited ability to evaluate the relative impact of each me-Hematoxicity and Benzene 245

tabolite on hematologic outcomes. Catechol was, however, more strongly associated with the MCV, a measure of RBC toxicity, and hydroquinone was slightly more strongly correlated with the absolute lymphocyte count. No combination of metabolites was more strongly associated with peripheral blood cells than individual metabolites. Individual benzene air levels 

### in these factories were much higher than we had expected to find, based 

### on historical area monitoring data. As a direct result 

### of our study, remedial action was taken at the two workplaces with the highest benzene exposures,including substitution oftoluene forbenzene, enclosure 

### ofreaction vessels, and improvement 

### in ventilation. Exposurepatterns 

### in the three factories evaluated 

### in this study are not representative of general exposure patterns 

### in China today. Based on data from 680 factories in 12cities in China (Dosemeciet al., 19941,average exposure was 33 ppm from 1965 to 1969. decreasing to 8 ppm by 1985-1987. Recent exposure data collected from workplaces 

### in Shanghai indicate that benzene levels have continued to decline 

### in many factories up tothe present time (Wang, 

### Y.Z..unpublished data). Current exposure 

### to benzene 

### in the United States is regulated at 1 ppm as an 8-hr TWA with a 15-min shortterm exposurelimit of 

### 5 ppm [OSHA. 19871.Although this level of benzene exposure is not considered hematotoxic, acute high-dose exposures are still being reported in the United States [e.g.. Midzenski et al., 19921. Our results support Goldstein’s recommendation that close attention should be paid to the absolute lymphocyte count when monitoring workers for early biologic effects due to benzene exposure [Goldstein, 19881.Although small changes 

### in the absolute lymphocyte count are unlikely to have any shortterm clinical consequences per se, this readily measurable and inexpensive marker may identify occupational environments that place workers at particularly increased risk 

### for subsequent development of hematopoietic malignancies. LaMontagne and colleagues [19931have reviewed the
use 

### of the complete blood count with differential as a screening tool. They note that automated differential cell countersare likely to eliminate many of the sources 

### of error present in manually performed WBC differentials, which
are generally performed on only 100 WBCs (also commented on by Goldstein, 1988).They recommend that at least 400WBCs be evaluated when manual counts are necessary. In our study. the automated absolute lymphocyte count (~10~ 

### pL) was significantly lower among workers 

### in the 131 pprn exposure group compared to controls (mean [sd] 1.6[0.3] vs. 1.9 [0.4],p =0.006, 16% decrease),while the absolute lymphocyte count derived from a standard manual count 

### of 100cells was similar among exposed and control subjects (I

### .6 

### [0.6]vs. 

### 1.7 

### [0.5], p 

### = 

### 0.55). Finally, recent studies suggest that benzene may alter patterns of particular lymphocyte subsets [Zeman et al., 1990; Luan, 19921.It is warranted to further explore these findings at current levels of benzene exposure,particularly since very accurate lymphocyte subset analysis by flow cytornetry has become widely available 

### in the United States.


### ACKNOWLEDGMENTS

We gratefully acknowledge the participation of the study subjects and the assistance of staff at 

### the Chinese Academy of Preventive Medicine. Beijing, China, and the Shanghai Hygiene and Anti-Epidemic Center. Shanghai, China.Thisstudy was supported

### inpart by the U.S. National Cancer Institute Epidemiology and Biostatistics program, contract DE-AC04-76EV01013 fromthe

### U.S. Department of Energy, Office of Health and Environmental Research (W.E.B.)and grants P42ES04705 and P30ES01896 fromthe National Institute of Environmental Health Sciences (M.T.S.). 

### L.-P.Z. is a trainee 

### of the Health Effects Component of the University of California Toxic Substances Program. 

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