AMERICAN JOURNAL OF INDUSTRIAL MEDICINE 29:236-246 (1 996) 

Hematotoxocity Among Chinese Workers 

Heavily Exposed to Benzene 

Nathaniel Rothman, MD, MPH, MHS, Gui-Lin Li, MD, Mustafa Dosemeci, PhD, 

William E. Bechtold, PhD, Gerald E. Marti, MD, PhD, Yao-Zu Wang, MD, 

Martha Linet, MD, Li-qiang Xi, MD, Wei Lu, MD, Martyn T. Smith, PhD, 

Nina Titenko-Holland, PhD, Luo-Ping Zhang, PhD, William Blot, PhD, 

Song-Nian Yin, MD, and Richard B. Hayes, PhD 

KEY WORDS: benzene, hematotoxicity, phenol, rnuconic acid, Itydroquinone, calechol 

Abbreviations: GUMS, gas chromatography/mass spectroscopy: WBC. white 

blood cell; MCV, mean corpuscular volume; TWA, time-weighted average; ppm, 

parts per million: SHAC, Shanghai Hygiene and Anti-Epidemic Institute; NCI. 

National Cancer Institute; RBC, red blood cell. 

Epidemiology and Biostatistics Program, National Cancer Institute, Bethesda, 

MD (N R.. M.D.. M.L.. W.BI.. R.B.H.). 

Chinese Academy of Preventive Medicine, Institute of Occupational Medicine, 

Beijing, China (G.-L.L.. L.-Q.X., S.Y.). 

Lovelace Inhalation Toxicology 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 

University of California, School of Public Health, Berkeley, CA (M.T.S, N.T.-H., 

Address reprint requests to Dr. Nathaniel Rothman, NlHlNCl EPN 41 8, BeAccepted for publication March 17, 1995. 

Health, Shanghai, China (Y.-Z.W.. W.L.). 

L.-P.Z.). 

thesda, MD 20892. 

(C) 1996 Wiley-Liss, Inc. 'This article is a US Government work and, as 

such, is in the public domain in the 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. 

MATERIALS AND METHODS 

Factory Identification and 

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 exposures to 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 to toluene. 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 chemicals associated 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 exposure to other known 

marrow-toxic chemicals or ionizing radiation. Eligibility 

criteria for controls were no history of occupational exposure to benzene. other marrow-toxic chemicals. 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 for benzene, 

toluene. and xylene. An 8-hr TWA 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 Clayton Environmental 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 changed the 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. 

The majority of subjects in the control factory that manufactured sewing machines were monitored for air levels of 

benzene. toluene, and xylene for I day on any of 6 different 

sampling days. 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 assess historical exposure in the cohort study [Dosemeci et 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 I lec t i on 

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 TS40 blood 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 (x 103/pL blood), the hematocrit (%). the red blood cell count (RBC; xlOh/pL blood), 

and the platelet count (x103/pL blood). The mean corpuscular volume (MCV; pm') was calculated as the hematocriv 

(RBC count x 10). The WBC 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 

100 x I O'/pL blood in both 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 pL of urine samples, followed by 100 pL 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 extracted with 2 niL of ethyl acetate. The ethyl 

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 100 pL 

of bistriniethylsilytrifluoracetamide, incubated at 60°C for 

30 min. and analyzed by GC/MS. 

Statistical Met hods 

Arithmetic mean L- standard deviation (ad) and median 

with range are used 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 f 7.3 

Females (n=21) 0.0 0.0 

Cigarettes/day 

Males (n=23) 11 .O f 7.8 13.5 f 13.7 

Drinks of alcohollweek 1.5 k 3.2 1.4 f 2.2 

Quetelet's index (weight,,/height,,,,,,2) 21.6 f 3.2 21.5 f 2.3 

uous outcome data that were not normally distributed were 

logarithmically transformed to improve normality. Analysis 

of covariance was 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-sided p values were calculated throughout: p values < 

0.05 were 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 comparable between exposed and control subjects (Table I). Twenty-one of 44 (48%) subjects in each group were 

female. 

Exposure Assessment 

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-hr TWA benzene personal air level was 3 1 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 subjects monitored 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 exposure value (3 I ppm) compared to those exposed to 

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 >3 1 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 [ 1.91 x I03/pL vs. 6.8 

[ 1.71 x 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 pm3 in 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 in exposed women 

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 [ 1.81 vs. 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 in 
240 Rothman et al. 

TABLE 11. Current Benzene. Toluene, and Xylene 8-Hr TWA 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 (0 01-0 1) 0 02 (0 02-0 3) 0 02 (0 02-0 6) 

Exposed 

5 31 ppm (n = 22)’ 

> 31 ppm (n = 22) 

13 6 (1 6-30 6) 

91 9 (31 5-328 5) 

0 02 (0 01-9 7) 0 02 (0 02-1 2 8) 

0 1 (0 02-12 9) 0 2 (0 03-7 2) 

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 Exposure Category, 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 (3 7-55 3) 3 2 (1 6-8 7) 0 18 (0 1-0 8) 1 6 (0 9-6 7) 

Exposed 

531 ppm (n = 22)b 

>31 pprn (n = 22)c 

38 9 (15 2-217 1) 

349 7 (36 8-516 7) 

7 0 (2 5-38 9) 

65 7 (5 0-84 2) 

8 15 (2 3-55 5) 

46 8 (8 6-103 4) 

12 8 (3 2-61) 

64 3 (9 7-196 6) 

‘Units pg/mg creatinine for all metabolites 

“A subsarnple 01 17 controls 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 provided a 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 lymphocyte count (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 to 13 I pptn benzene. These group differences were 

niininially changed after adjustment for alcohol consumplion, cigarctte use, and Quelelet‘s index. 

Sirice the lower exposed group (131 ppm) contained 

some subjects with 8-hr TWA exposures above 3 I 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 to the benzene-exposed workers, we examined the strength of the exposure-response relationship over the entire range of exposures 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 of benzene, and metabolism. Only 

the total WBC count, absolute lymphocyte count, and MCV 

showed evidence for 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 

(Table V), 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). 
Hematoxicity and 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 k sd Mean f sd Mean f sd Mean f sd lean ? sd Mean t sd 

(range) (range) (range) (range) (range) (range) 

Control (n = 44) 6.8 ir 1.7 1.9 t 0.4 4.7 f 0.6 42.0 t 5.6 88.9 f 4.9 166k 59 

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 f 1.8 1.6 f 0.3 4.6 k 0.6 41.2 t 5.7 89.8 f 3.9 132 f 45 

(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 f 1.9 1.3 t 0.3 4.2 f 0.6 38.8 f 5.3 92.9 f 3.4 121 f 43 

(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 

The proportion of granulocytes in the WBC differential 

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 

0.58 to 0.1%. The percent monocytes and 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 granulocytes times the total WBC count 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 group to 

4.0 (1.8) among workers in the higher exposure group (p = 

0.28). whereas the absolute lymphocyte count (x 1 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). These differences were minimally changed after 

adjustment for alcohol consumption, cigarette use, and 

Quetelet's index. 

DISCUSSION 

Forty-four workers exposed to a wide range of benzene 

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 = 7.6 ppm (range 1-20 ppm) 

compared to controls. Other hematologic parameters were 

altered in the exposed group compared to controls and are 

consistent with hematotoxic effects of benzene reviewed by 

Aksoy [ 19881 and Goldstein [ 19771, i.e., toxicity for WBC, 

platelets. RBC, and a tendency towards macrocytosis. However. in our study, none of these measures was as sensitive 

an indicator of benzene exposure as the absolute lymphocyte count, particularly at levels 131 ppm. 

Among the benzene-exposed workers, only the WBC 

count, absolute lymphocyte count, and MCV were significantly correlated with continuous (vs. categorized) measures of benzene exposure. The finding of overall decreases 

among benzene-exposed workers of other peripheral blood 

cells, and yet the lack of a dose-response relationship, may 

reflect a more idiosyncratic reaction to benzene exposure. In 

addition, in morphologic studies. we found that only RBC 

abnormalities, and not those of any type of WBC or platelet, 

were associated with benzene exposure, and only among the 

most heavily exposed workers. 

Goldwater [ 194 I ] noted that lymphocytopenia was 

more frequently found among subjects exposed to benzene 

than was leukopenia. Aksoy found that 28 of 37 leukopenic 
242 Rothman et al. 

I 

2.8 - 

2.6 

2.4 

I - 

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, in a study of workers exposed to 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 measures for 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 range of remaining data except for 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 results of animal 

studies demonstrating sensitivity of lymphatic tissue to henzene [Snyder et al., 1980; Ward et al., 19851 at concentrations as low as I0 ppm benmie [Buarson et al., 19831. with 

numerous reports that have described an excess of chrornosome damage in lymphocytes from workers exposed to ben- 
Hematoxicity and Benzene 243 

loo i 

98 - 

96 

94 - 

I 

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) 

FIGURE 2. Red blood cell mean corpuscular volume, by current benzene exposure category, in a study of 

workers exposed to benzene in Shanghai, China, 1992. Symbols as for Fig. 1 ., plus *extreme outlier defined 

as >3.0 x 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 of workers exposed to 

benzene and unexposed controls have not reported data on 

the absolute lymphocyte count, including studies of workers 

exposed up to 1.4 ppm [Collins et al.. 19911. 10 ppm [Yardley-Jones et al.. 19881. 12.4 ppm [Sarto et al.. 19841, 31.5 

ppm [Hancock et al., 19841, and 35 ppm benzene 

[Townsend et al., 19781. These authors generally 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., 19881 or small declines in the RBC count [Townsend et 

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 Counts With 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 0 00 030 -002 

Muconic (n=43) -0 23 -0 30 -0 11 0 00 0 3Za -0 0006 

Hydroquinone (n = 43) -0 26 -0 36a - 0 084 0 00 0 36a 0 016 

Catechol (n = 42) -0 28 -0 25 -0 14 0 00 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 from a linear regression model 

'p < 005 

bp < 0 001 

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/ a bnormall 

total total 

Control (n = 42) 3/42 (7%) 7/40 (1 7%) 

Exposed 

531 ppm (n = 22) 1/20 (5%) 2/21 (10%) 

Platelet 

abnormality 

No. 

a bnormall 

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 23 I 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 effects of 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 shown to he ii more potent inducer 

of sister chromatid exchange than hydroquinone [Erexson et 

al., 1985; Glatt et al., 1989; Morimoto et al., 19931. Further, 

phenol [Subrahmanyam et al., 19901 has been shown to 

stimulate hydroquinone bioactivation and potentiate hydroquinone-induced myelotoxicity (Eastinond et al.. 19871, and 

catechol has been shown to stimulate hydroquinone bioactivation [Subrahmanyam et al., 19911 and potentiate hydroquinone-induced micronuclei formation [Robertson et al., 

199 1 J. Thus, the complex nature of benzene metabolite interactions precludes tirm conclusions about the relevance of 

alterations in benzene 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 of toluene 

for benzene, enclosure of reaction vessels, and improvement 

in ventilation. Exposure patterns 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 12 cities in China (Dosemeci et 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 to the 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 exposure limit 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 [ 19931 have reviewed the 

use of the complete blood count with differential as a 

screening tool. They note that automated differential cell 

counters are 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 400 WBCs 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 100 cells 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. This study was supported in part by the U.S. National 

Cancer Institute Epidemiology and Biostatistics program, 

contract DE-AC04-76EV01013 from the U.S. Department of 

Energy, Office of Health and Environmental Research 

(W.E.B.) and grants P42ES04705 and P30ES01896 from the 

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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