Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Selenium status in workers handling aromatic nitro‐amino compounds in a chemical factory Munehiro Yoshida , Masahiko Sunaga & Ichiro Hara To cite this article: Munehiro Yoshida , Masahiko Sunaga & Ichiro Hara (1990) Selenium status in workers handling aromatic nitro‐amino compounds in a chemical factory, Journal of Toxicology and Environmental Health, 31:1, 1-10, DOI: 10.1080/15287399009531433 To link to this article: http://dx.doi.org/10.1080/15287399009531433
Published online: 20 Oct 2009.
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Date: 13 November 2015, At: 23:08
SELENIUM STATUS IN WORKERS HANDLING AROMATIC NITRO-AMINO COMPOUNDS IN A CHEMICAL FACTORY Munehiro Yoshida, Masahiko Sunaga, Ichiro Hara
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Department of Public Health, Kansai Medical University, Moriguchi Osaka, Japan
The selenium status of workers handling aromatic nitro-amino (ANA) compounds was evaluated by measurement of their blood and urinary selenium concentrations and blood glutathione peroxidase (CSH-Px) activities. Forty-seven healthy Japanese male workers (42.7 ± 12.1 yr) handling ANA compounds routinely in a chemical factory were studied as exposed workers, and 107 nonindustrial healthy Japanese males (39.3 ± 10.0 yr) in the same region served as a control group. Urinary diazoreactionpositive metabolites and methemoglobin, both of which have been used as indices of exposure to ANA compounds, were significantly elevated in the exposed workers. Both plasma and erythrocyte selenium in the exposed workers showed 20% lower values compared to the control group. GSH-Px activities in plasma and erythrocytes were also significantly decreased in the exposed workers, but urinary selenium excretions were similar between the two groups. Questionnaire information obtained from each subject regarding intake habits of selenium-rich foods (bread, eggs, meat and fish) indicated that the average dietary selenium intake was similar for the control group and the exposed workers. These results indicate that (1) the workers handling ANA compounds were surely exposed to these chemicals; (2) their selenium status was lower than that of the nonindustrial controls; and (3) the low selenium status was not associated with any dietary factor.
INTRODUCTION Recent epidemiological studies have suggested a relation between the incidence of certain types of cancer (Shamberger et al., 1976; SaIonen et al., 1984) or cardiovascular disease (Salonen et al., 1982; Kok et al., 1989) and selenium status. To evaluate selenium status, blood selenium levels and glutathione peroxidase (GSH-Px) activities have been measured in a variety of groups of healthy subjects from several different geographical regions (Thomson et al., 1977; Whanger et al., 1988). Geographic comparisons of blood selenium levels or GSH-Px activities have demonstrated a distinct variation in selenium status, which correlates with the dietary selenium intake (Combs and Combs, 1986). This study was supported by scientific grant in aid no. 01770374 for M. Yoshida from the Ministry of Education, Science and Culture of Japan. Requests for reprints should be sent to Munehiro Yoshida, Department of Public Health, Kansai Medical University, Fumizono-cho Moriguchi Osaka 570, Japan.
Journal of Toxicology and Environmental Health, 31:1-10, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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M. YOSHIDA ET AL.
Other than selenium intake, smoking habits (Ellis et al., 1984) or working in a particular industrial setting (Lane et al v 1983; Zachara et al., 1987) affects selenium status. Changes in selenium status have been found to affect the toxicities of several xenobiotics (Combs and Combs, 1987). Thus, for people exposed to chemical materials occupationally, selenium status may be an important factor in preserving their health, especially in low selenium areas. Aromatic nitro-amino (ANA) compounds are used as raw materials in the manufacture of dyes, rubber, pesticides, and drugs. A common toxicological reaction to these compounds is the oxidation of hemoglobin (Hb) to methemoglobin (MetHb) followed by cyanosis and anemia (Beard and Noe, 1981). In the present study, in order to assess the selenium status of people exposed to ANA compounds, we measured blood and urinary selenium concentrations and blood GSH-Px activities of workers handling ANA compounds routinely in a chemical factory. SUBJECTS AND METHODS Subjects
Forty-seven healthy male Japanese workers in a chemical factory in Osaka prefecture, aged 20-64 (mean ± SD, 42.7 ± 12.1) yr, were studied as exposed workers. They worked the day shift (8 a.m. to 4 p.m.) in a 5-d work week. In this factory, the workers routinely handle ANA compounds, including p-aminophenol, oaminophenol, p-anisidine, pnitroaniline, p-chloronitrobenzene, o-chloronitrobenzene, and 4-chloro2-aminophenol, as raw materials for the production of dyes or drugs. For the past two decades, the yearly amounts of ANA compounds handled have remained almost constant. The main duties of the workers included putting the ANA compounds from paper bags into reaction vessels, taking out reaction products from the vessels, and bagging the reaction products into paper bags. A few workers carried the paper bags by folklifts to the warehouse. The workers wore fabric protective clothing and cotton gloves during these duties. Only when the workers were engaged in bagging did they wear particulate-filter respirators and polyvinyl chloride coated cotton gloves. One hundred and seven healthy nonindustrial Japanese males, aged 22-62 (mean ± SD, 39.3 ± 10.0) yr, served as a control group. This group consisted of office workers and drivers in companties that did not handle any chemical compounds and a few teaching staff of our university. They live in the same geographic area as the exposed workers. All the subjects were examined for serum alanine aminotransferase; aspartate aminotransferase; 7-glutamyltranspeptidase; albumin; as well as urine glucose, protein, and blood; and people suspected of having a dysfunction of the liver or kidneys or malnutrition were excluded from
SELENIUM STATUS I N CHEMICAL FACTORY WORKERS
the study. Questionnaire information obtained from each subject regarding smoking and drinking habits indicated that the consumption of cigarettes and alcoholic drinks was similar for the control group and the exposed workers. The information was as follows (means ± SD): smoking habits (cigarettes/d), 16.1 ± 12.0 (control) and 13.9 ± 11.1 (exposed); consumption of alcoholic drinks (ml ethanol/d), 32.4 ± 27.7 (control) and 26.9 ± 24.7 (exposed).
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Blood and Urine Sampling
Blood samples were taken into heparinized tubes by a cubital venipuncture. Within 4 h after the sampling, hematocrit (Ht), Hb, and MetHb were determined and the blood was centrifuged. Plasma was collected and the erythrocytes thus obtained were washed three times with an excess of saline. The erythrocytes were hemolyzed by 4 volumes of a hypotonie phosphate buffer (5 mJW, pH 7.0). Selenium and GSH-Px were assayed for the plasma and hemolysate. After the assay of GSH-Px within 2 d, the plasma and hemolysate were frozen at — 20°C and kept until the selenium analysis. Urine samples were taken into plastic tubes. Creatinine, diazoreaction-positive substances (DRPS), and selenium levels were determined. The urine was kept frozen at -20°C until the analyses. The blood and urine sampling was performed from 9 to 11 a.m. on mornings in November and December 1988. Evaluation of Selenium-Rich Food Intake
Since the main selenium sources of the Japanese diet are wheat products, eggs, meat, and fish (Suzuki et al., 1988), dietary selenium intake can be estimated by obtaining information about the intake habit of these foodstuffs. Weekly intake of bread, eggs, meat, and fish was assessed by a simplified questionnaire as described previously (Yoshida, 1990). Assays GSH-Px activity in the plasma and hemolysate was assayed by a modified method of Paglia and Valentine (1967) using 0.29 mW ierf-butyl hydroperoxide as the peroxide substrate (Yoshida et al., 1981). Selenium determination was performed fluorometrically by the method of Watkinson (1966). To ensure the accuracy of selenium determination, NBS bovine liver (NBS-SRM 1577a) was simultaneously measured. MetHb level was measured spectrometrically by the method of Hasegawa et al. (1962). Urinary DRPS was determined by the method of Watanabe et al. (1976) with p-aminophenol as the standard material. Hb and urinary creatinine were measured by the use of commercial kits: Hb, Hemoglobin B-Test
M. YOSHIDA ET AL.
(Wako Pure Chemical Industries Ltd., Osaka); creatinine, Creatinine-Test (Wako). Statistics
Data were evaluated by Student's i-test. The results for the urinary DRPS formed a log-normal distribution, so these data were logarithmically transformed before the statistical analysis.
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RESULTS Levels of Exposure to ANA Compounds
Levels of urinary DRPS and hematological values are shown in Table 1. Urinary DRPS levels of the exposed workers were significantly (p < .001) higher than those of the control group. Because in vivo metabolites of ANA compounds show a positive reaction in the diazo reaction, and urinary DRPS has been utilized as an index of exposure to ANA compounds (Yoshida et al., 1988), the increase of urinary DRPS indicates that the workers in the present study were surely exposed to ANA compounds. In response to the exposure, MetHb levels of the workers were elevated to more than twice those of the control group. Both Ht and Hb values were within the normal ranges in both groups, but were significantly decreased in the exposed workers. Selenium Status
Selenium concentrations and GSH-Px activities in the plasma and erythrocytes are summarized in Table 2. Both plasma and erythrocyte selenium in the exposed workers showed 20% lower values compared to TABLE 1. Urinary Diazo-Reaction-Positive Metabolites and Hematological Values
Urinary diazo metabolites (g p-aminophenol equivalent/ g creatinine)a Hemoglobin (g/100 ml) Hematocrit (%) Methemoglobin (% to Hb)
Control group (n = 107)
Exposed workers (n - 47)
0.33 15.3 45.8 0.5
0.47 (0.20-1.12)b 14.7 ± 0.96 44.5 ± 2.6C 1.2 ± 0.5b
(0.23-0.49) ± 0.9 ± 2.5 ± 0.3
Note. Values except urinary diazo metabolites are means ± SD. Values are geometrical means with SD ranges in the parentheses. ^Significant difference was observed between the control and exposed groups by Student's t-test at p < .001. Significant at p < .05. a
SELENIUM STATUS I N CHEMICAL FACTORY WORKERS
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TABLE 2. Blood and Urinary Selenium Concentrations and Blood Glutathione Peroxidase Activities
Selenium concentration Plasma (ng/ml) Erythrocytes (ng/ml packed cell) Erythrocytes (pg/g Hb) Urine (/¿g/g creatinine) Clutathione peroxidase activity3 Plasma (unit/ml) Erythrocytes (unit/g Hb)
Control group (n " 107)
(n - 47)
118 222 0.67 29
96 177 0.54 27
± ± ± ±
25 60 0.18 12
0.26 ± 0.05 19.0 ± 5.3
Exposed workers
± ± ± ±
166 54b 0.16b 10
0.19 ± 0.046 16.5 ± 3.9C
Note. Values are means ± SD. One unit of glutathione peroxidase activity is expressed as 1 /¿mol of NADPH oxidized per minute. ^Significant difference was observed between the control and exposed groups by Student's f-test a t p < .001. Significant at p < .01. a
those in the control group. Similar to the selenium, the GSH-Px activities were significantly decreased in the exposed workers, but the extent of decrease was different between the plasma and erythrocytes; the plasma GSH-Px was 27% decreased, whereas the erythrocyte enzyme was 13% decreased. In Table 2, urinary selenium excretions are also presented. Unlike the blood selenium, urinary selenium levels in the exposed workers were similar to those in the control group. Habit of Selenium-Rich Food Intake
Table 3 shows weekly intakes of bread, eggs, meat, and fish assessed by the questionnaire. Differences between the exposed workers and the control group were not observed in weekly intakes of the four seleniumTABLE 3. Habits of Bread, Eggs, Meat, and Fish Intake
Bread (slices of white bread/week)a Eggs (number/week) Meat (frequency/week)b Fish (frequency/week)6
Control group (n = 107)
Exposed group (n = 47)
4.8 4.9 5.2 5.1
5.4 5.2 4.7 5.0
± ± ± ±
3.5 2.3 2.5 2.7
± ± ± ±
3.6 2.9 2.8 2.4
Nofe. Values are means + SD. One piece of any other type of bread was regarded as being equivalent to one slice of white bread. frequency of intake at lunch and supper. a
M. YOSHIDAETAL.
rich foods. This indicates a similarity between the two groups in average daily selenium intake.
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DISCUSSION
Generally, the toxic effect of ANA compounds is evaluated by the ability to form MetHb (Watanabe et al., 1976). As a control of the industrial use of MetHb-producing materials such as ANA compounds, the American Conference of Governmental Industrial Hygienists (1988) has proposed a value of 1.5% MetHb as a biological exposure index (BEI), which is a reference value intended as a guideline for the evaluation of potential health hazards, including any chronic effects. In the present study, the workers engaged in the production of dyes or drugs in the chemical factory were surely exposed to ANA compounds. In response to this exposure, the MetHb levels of the workers was apparently increased, and the levels exceeded the BEI in some workers. Duration of the handling of ANA compounds in the factory was 0.7-31.5 (mean ± SD, 14.0 ± 8.7) yr and the amounts of ANA compounds handled remained almost constant over the past two decades; the formation of MetHb at a level around the BEI has therefore continued for a long time in the workers. Long-term methemoglobinemia is considered to result in an anemia; in fact, Hb and Ht values were decreased in the exposed workers. Compared to the control group, blood selenium concentrations and GHS-Px activities in the exposed workers were significantly decreased; selenium status in the exposed workers was therefore apparently lowered. However, careful verification is necessary before the conclusion is drawn that the low selenium status in the workers was associated with occupational exposure to ANA compounds. Dietary selenium intake is the most important factor to contribute to the variation of the selenium status of populations in different geographical areas (Combs and Combs, 1986). As for healthy Japanese populations in a common geographical area, the erythrocyte selenium levels are significantly correlated with the habit of fish intake (Suzuki et al., 1989; Yoshida, 1990). However, as described in Table 3, the intake habits of the four selenium-rich foods including fish were similar for the exposed workers and the controls; average dietary selenium intake is therefore considered to be similar in the two groups. Suzuki et al. (1989) reported that plasma selenium levels were significantly correlated with the selenium intake from wheat products in the diets of subjects prior to blood sampling. On the other hand, we previously observed that urinary selenium excretion before noon was increased in subjects who consumed a large amount of selenium-rich foods at breakfast on the day of the urine sampling and at supper on the preceding day (Yoshida, 1990). In the present study, urinary sele-
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SELENIUM STATUS IN CHEMICAL FACTORY WORKERS
nium levels were similar in the two groups. Although we did not determine the contents of diet before the blood or urine sampling, a similarity in the selenium intake before the sampling between the two groups may exist on the assumption that the rate of selenium excretion is not altered in the exposed workers. Other than the selenium intake, several factors have been known to influence the selenium status in healthy populations; these factors include age (Miller et al., 1983), smoking habits (Ellis et al., 1984), and alcohol abuse (Lloyd et al., 1983). However, as described earlier, differences in these factors did not exist between the exposed and control groups. Accordingly, it is reasonable to consider that the low selenium status in the exposed workers was associated with working at the chemical factory. There have been few studies regarding the selenium status of industrial workers. Lane et al. (1983) reported that selenium concentrations of plasma and erythrocytes and GSH-Px activities of erythrocytes in oil refinery workers in Texas were significantly lower than those in controls in the same region. Similar low selenium status, including low blood and urinary selenium and blood GSH-Px, was also reported in power station and rubber factory workers in Poland (Zachara et al., 1987). In these studies, two explanations for the low selenium status have been proposed: (1) Selenium may be lost in perspiration. (2) A chemical oxidant stress in the occupational environments may influence the distribution and utilization of selenium: This relationship has been suggested on the basis of some animal experiments in which environmental hazards such as polychlorinated biphenyls and organic solvents such as carbon tetrachloride produced tissue peroxidation and exacerbation of selenium deficiency symptoms (Combs and Scott, 1975; Hafeman and Hoekstra, 1977). The present workers must surely have perspired greatly, because the temperature in the working room of the factory was more than 30°C, even in December. Moreover, ANA compounds and their in vivo metabolites have oxidative stress properties such as the formation of MetHb (Beard and Noe, 1981). Thus both proposed explanations seem to be applicable to the present study. However, Levander et al. (1981) pointed out that selenium excretion into sweat occurred only in small amounts, and, unlike the Polish study, the urinary selenium levels were not decreased in the workers. Thus, we consider that only the explanation of a chemical oxidant stress is appropriate for the low selenium status of the exposed workers; that is, the low selenium status was associated with the exposure to ANA compounds. Another possible explanation for the low selenium status in the exposed workers is that the ANA compounds were metabolized to GSH conjugates (Beard and Noe, 1981) and may have affected GSH metabolism (Kosower and Kosower, 1978). In animal experiments, selenium sta-
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M. YOSHIDA ET AL.
tus influences GSH metabolism (Hill and Burk, 1982; Yoshida et al., 1984), and inversely, selenium metabolism is connected with GSH (Hsieh and Ganther, 1971). The low selenium status in the exposed workers may be caused via an altered GSH status. In the present study, not only the selenium levels but also GSH-Px activities were decreased in the exposed workers. According to the New Zealand studies, there is an excellent correlation between selenium levels and GSH-Px activities in whole blood at concentrations below about 0.10 /¿g/ml, or in erythrocytes at concentrations below 0.14 /¿g/ml (Thomson et al., 1977; Rea et al., 1979). Above these concentrations, GSH-Px activity is not correlated with selenium level. Most of the present subjects showed concentrations above these values. Thus, the low GSH-Px activities in the exposed workers may not be directly caused by the low selenium levels. The factor causing the low GSH-Px activities may differ from the factor producing the low selenium levels; there may not be a single cause of the low selenium status. Animal research shows that low selenium status elevates the toxicity of some xenobiotics (Peterson et al., 1982; Combs and Peterson, 1983). Moreover, susceptibility to oxidant stress of erythrocytes, involving hemolysis or MetHb formation, is increased by low selenium status (Rotruck et al., 1972; Kim et al., 1988). Thus, attention must be paid to the low selenium status observed in workers handling ANA compounds. REFERENCES American Conference of Governmental Industrial Hygienists. 1988. Threshold Limit Values and Biological Exposure Indices for 1988-1989, pp. 60-61. Cincinnati: American Conference of Governmental Industrial Hygienists. Beard, R. R., and Noe, J. T. 1981. Aromatic nitro and amino compounds. In Patty's Industrial Hygiene and Toxicology, 3rd rev. ed., eds. G. D. Clayton and F. E. Clayton, vol. 2A, pp. 24132489. New York: Wiley and Sons. Combs, G. F., Jr., and Combs, S. B. 1986. The Role of Selenium in Nutrition, pp. 327-399. New York: Academic Press. Combs, G. F., Jr., and Combs, S. B. 1987. Selenium effects on drug and foreign compound toxicity. Pharmac. Ther. 33:303-315. Combs, G. F., Jr., and Peterson, F. J. 1983. Protection against acute paraquat toxicity by dietary selenium in the chick. J. Nutr. 113:538-545. Combs, G. F., Jr., and Scott, M. L. 1975. Polychlorinated biphenyl-stimulated selenium deficiency in the chick. Poult. Sci. 54:1152-1158. Ellis, N., Lloyd, B., Lloyd, R. S., and Clayton, B. E. 1984. Selenium and vitamin E in relation to risk factors for coronary heart disease. J. Clin. Pathol. 37:200-206. Hafeman, D. G., and Hoekstra, W. G. 1977. Protection against carbon tetrachloride-induced lipid peroxidation in the rat by dietary vitamin E, selenium and measured by ethane evolution. J. Nutr. 107:656-665. Hasegawa, H., Sato, M., Yoshikawa, H., Sakabe, H., Yamaguchi, M., and Hotta, K. 1962. Nitroglycol poisoning in an explosive plant (II). Bull. Natl. Inst. Ind. Health 5:10-20. Hill, K. E., and Burk, R. F. 1982. Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes. J. Biol. Chem. 257:10668-10672.
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Hsieh, H. S., and Canther, H. E. 1971. Acid-volatile selenium formation catalyzed by glutathione reductase. Biochemistry 14:1632-1636. Kim, C-H., Yasumoto, K., Suzuki, T., and Yoshida, M. 1988. Failure of glutathione in suppressing tert-butyl hydroperoxide-induced hemolysis in vitro of selenium-deficient rat erythrocytes. Nutr. Res. 8:767-775. Kok, F. J., Hofman, A., Witteman, J. C. M., de Bruijn, A. M., Kruyssen, D. C. M., de Bruin, M., and Valkenburg, H. A. 1989. Decreased selenium levels in acute myocardial infarction. J. Am. Med. Assoc. 261:1161-1164. Kosower, N. S., and Kosower, E. M. 1978. The glutathione status of cells. Int. Rev. Cytol. 54:109160. Lane, H. W., Warren, D. C., Martin, E., and McCowan, J. 1983. Selenium status of industrial worker. Nutr. Res. 3:805-817. Levander, O. A., Sutherland, B., Morris, V. C., and King, J. C. 1981. Selenium metabolism in human nutrition. In Selenium in Biology and Medicine, 2nd ed., eds. J. E. Spallholz and H. E. Canther, pp. 256-268. Westport, Conn.: AVI. Lloyd, B., Lloyd, R. S., and Clayton, B. E. 1983. Effect of age, smoking, alcohol, and other factors on the selenium status of a healthy population. J. Epidemiol. Community Health 37:213-217. Miller, L., Millis, B. J., Blotcky, A. J., and Lindeman, R. D. 1983. Red blood cell and serum selenium concentrations as influenced by age and selected disease. J. Am. Coll. Nutr. 4:331-341. Paglia, D. E., and Valentine, W. N. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169. Peterson, F. J., Combs, C. F., Jr., Holtzman, J. L., and Mason, R. P. 1982. Effect of selenium and vitamin E deficiency on nitrofurantoin toxicity in the chick. J. Nutr. 112:1741-1746. Rea, H. M., Thomson, C. D., Campbell, D. R., and Robinson, M. F. 1979. Relation between erythrocyte selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities of New Zealand residents and visitors to New Zealand. Br. J. Nutr. 42:201-208. Rotruck, J. T., Pope, A. L., Canther, H. E., and Hoekstra, W. C. 1972. Prevention of oxidative damage to rat erythrocytes by dietary selenium. J. Nutr. 102:689-696. Salonen, J. T., Alfthan, C., Huttunen, J. K., Pikkarainen, J., and Puska, P. 1982. Association between cardiovascular death and myocardial infarction and serum selenium in a matchedpair longitudinal study. Lancet 2:175-179. Salonen, J. T., Alfthan, G., Huttunen, J. K., and Puska, P. 1984. Association between serum selenium and the risk of cancer. Am. J. Epidemiol. 120:342-349. Shamberger, R. J., Tytko, S. A., and Willis, C. E. 1976. Antioxidant and cancer. Part IV. Selenium and age-adjusted cancer mortality. Arch. Environ. Health. 31:231-235. Suzuki, T., Imai, H., Kobayashi, K., Hongo, T., Kashiwazaki, H., Ohtsuka, R., Suzuki, H., and Ishida, H. 1988. Dietary intake of selenium in Japanese. An estimation by analyzed and reported values in foodstuffs and cooked dishes. Nippon Eiyo Shokuryo Cakkaishi (J. Jpn. Soc. Food Nutr. Sci.) 41:91-102. Suzuki, T., Hongo, T., Ohba, T., Kobayashi, K., Imai, H., Ishida, H., and Suzuki, H. 1989. The relation of dietary selenium to erythrocyte and plasma selenium concentrations in Japanese college women. Nutr. Res. 9:839-848. Thomson, C. D., Rea, H. M., Doesburg, V. M., and Robinson, M. F. 1977. Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents. Br. J. Nutr. 37:457-460. Watanabe, T., Ishihara, N., and Ikeda, M. 1976. Toxicity of and biological monitoring for 1,3diamino-2,4,6-trinitrobenzene and other nitro-amino derivatives of benzene and chlorobenzene. Int. Arch. Occup. Environ. Health 37:157-168. Watkinson, J. H. 1966. Fluorometric determination of selenium in biological material with 2,3diaminonaphthalene. Anal. Chem. 38:92-97. Whanger, P. D., Beilstein, M. A., Thomson, C. D., Robinson, M. F., and Howe, M. 1988. Blood selenium and glutathione peroxidase activity of population in New Zealand, Oregon, and South Dakota. FASEB J. 2:2996-3002. Yoshida, M. 1990. Relationship between dietary food consumption assessed by a simplified
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questionnaire and blood or urinary selenium concentration. Nippon Eiyo Shokuryo Gakkaishi (J.Jpn. Soc. Food Nutr. Sci.) 43:49-53. Yoshida, M., Yasumoto, K., Iwami, K., and Tashiro, H. 1981. Distribution of selenium in bovine milk and selenium deficiency in rats fed casein-based diets, monitored by lipid peroxide level and glutathione peroxidase activity. Agric. Biol. Chem. 45:1681-1688. Yoshida, M., Fukunaga, T., Iwami, K., and Yasumoto, K. 1984. Variation of glutathione level and synthesis activity in chick liver due to selenium and vitamin E deficiencies. J. Biochem. 96:1391-1397. Yoshida, M., Sunaga, M., and Hara, I. 1988. Urinary diazo-positive metabolites levels of workers handling p-nitrochIorobenzene in a dye producing factory. Ind. Health 26:87-91. Zachara, B. A., Wasowicz, W., Sklodowska, M., and Gromadzinska, J. 1987. Selenium status, lipid peroxides concentration, and glutathione peroxidase activity in the blood of power station and rubber factory workers. Arch. Environ. Health 42:223-229. Received October 21, 1989 Accepted March 30, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Selenium status in workers handling aromatic nitro‐amino compounds in a chemical factory Munehiro Yoshida , Masahiko Sunaga & Ichiro Hara To cite this article: Munehiro Yoshida , Masahiko Sunaga & Ichiro Hara (1990) Selenium status in workers handling aromatic nitro‐amino compounds in a chemical factory, Journal of Toxicology and Environmental Health, 31:1, 1-10, DOI: 10.1080/15287399009531433 To link to this article: http://dx.doi.org/10.1080/15287399009531433
Published online: 20 Oct 2009.
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View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 13 November 2015, At: 23:08
SELENIUM STATUS IN WORKERS HANDLING AROMATIC NITRO-AMINO COMPOUNDS IN A CHEMICAL FACTORY Munehiro Yoshida, Masahiko Sunaga, Ichiro Hara
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Department of Public Health, Kansai Medical University, Moriguchi Osaka, Japan
The selenium status of workers handling aromatic nitro-amino (ANA) compounds was evaluated by measurement of their blood and urinary selenium concentrations and blood glutathione peroxidase (CSH-Px) activities. Forty-seven healthy Japanese male workers (42.7 ± 12.1 yr) handling ANA compounds routinely in a chemical factory were studied as exposed workers, and 107 nonindustrial healthy Japanese males (39.3 ± 10.0 yr) in the same region served as a control group. Urinary diazoreactionpositive metabolites and methemoglobin, both of which have been used as indices of exposure to ANA compounds, were significantly elevated in the exposed workers. Both plasma and erythrocyte selenium in the exposed workers showed 20% lower values compared to the control group. GSH-Px activities in plasma and erythrocytes were also significantly decreased in the exposed workers, but urinary selenium excretions were similar between the two groups. Questionnaire information obtained from each subject regarding intake habits of selenium-rich foods (bread, eggs, meat and fish) indicated that the average dietary selenium intake was similar for the control group and the exposed workers. These results indicate that (1) the workers handling ANA compounds were surely exposed to these chemicals; (2) their selenium status was lower than that of the nonindustrial controls; and (3) the low selenium status was not associated with any dietary factor.
INTRODUCTION Recent epidemiological studies have suggested a relation between the incidence of certain types of cancer (Shamberger et al., 1976; SaIonen et al., 1984) or cardiovascular disease (Salonen et al., 1982; Kok et al., 1989) and selenium status. To evaluate selenium status, blood selenium levels and glutathione peroxidase (GSH-Px) activities have been measured in a variety of groups of healthy subjects from several different geographical regions (Thomson et al., 1977; Whanger et al., 1988). Geographic comparisons of blood selenium levels or GSH-Px activities have demonstrated a distinct variation in selenium status, which correlates with the dietary selenium intake (Combs and Combs, 1986). This study was supported by scientific grant in aid no. 01770374 for M. Yoshida from the Ministry of Education, Science and Culture of Japan. Requests for reprints should be sent to Munehiro Yoshida, Department of Public Health, Kansai Medical University, Fumizono-cho Moriguchi Osaka 570, Japan.
Journal of Toxicology and Environmental Health, 31:1-10, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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M. YOSHIDA ET AL.
Other than selenium intake, smoking habits (Ellis et al., 1984) or working in a particular industrial setting (Lane et al v 1983; Zachara et al., 1987) affects selenium status. Changes in selenium status have been found to affect the toxicities of several xenobiotics (Combs and Combs, 1987). Thus, for people exposed to chemical materials occupationally, selenium status may be an important factor in preserving their health, especially in low selenium areas. Aromatic nitro-amino (ANA) compounds are used as raw materials in the manufacture of dyes, rubber, pesticides, and drugs. A common toxicological reaction to these compounds is the oxidation of hemoglobin (Hb) to methemoglobin (MetHb) followed by cyanosis and anemia (Beard and Noe, 1981). In the present study, in order to assess the selenium status of people exposed to ANA compounds, we measured blood and urinary selenium concentrations and blood GSH-Px activities of workers handling ANA compounds routinely in a chemical factory. SUBJECTS AND METHODS Subjects
Forty-seven healthy male Japanese workers in a chemical factory in Osaka prefecture, aged 20-64 (mean ± SD, 42.7 ± 12.1) yr, were studied as exposed workers. They worked the day shift (8 a.m. to 4 p.m.) in a 5-d work week. In this factory, the workers routinely handle ANA compounds, including p-aminophenol, oaminophenol, p-anisidine, pnitroaniline, p-chloronitrobenzene, o-chloronitrobenzene, and 4-chloro2-aminophenol, as raw materials for the production of dyes or drugs. For the past two decades, the yearly amounts of ANA compounds handled have remained almost constant. The main duties of the workers included putting the ANA compounds from paper bags into reaction vessels, taking out reaction products from the vessels, and bagging the reaction products into paper bags. A few workers carried the paper bags by folklifts to the warehouse. The workers wore fabric protective clothing and cotton gloves during these duties. Only when the workers were engaged in bagging did they wear particulate-filter respirators and polyvinyl chloride coated cotton gloves. One hundred and seven healthy nonindustrial Japanese males, aged 22-62 (mean ± SD, 39.3 ± 10.0) yr, served as a control group. This group consisted of office workers and drivers in companties that did not handle any chemical compounds and a few teaching staff of our university. They live in the same geographic area as the exposed workers. All the subjects were examined for serum alanine aminotransferase; aspartate aminotransferase; 7-glutamyltranspeptidase; albumin; as well as urine glucose, protein, and blood; and people suspected of having a dysfunction of the liver or kidneys or malnutrition were excluded from
SELENIUM STATUS I N CHEMICAL FACTORY WORKERS
the study. Questionnaire information obtained from each subject regarding smoking and drinking habits indicated that the consumption of cigarettes and alcoholic drinks was similar for the control group and the exposed workers. The information was as follows (means ± SD): smoking habits (cigarettes/d), 16.1 ± 12.0 (control) and 13.9 ± 11.1 (exposed); consumption of alcoholic drinks (ml ethanol/d), 32.4 ± 27.7 (control) and 26.9 ± 24.7 (exposed).
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Blood and Urine Sampling
Blood samples were taken into heparinized tubes by a cubital venipuncture. Within 4 h after the sampling, hematocrit (Ht), Hb, and MetHb were determined and the blood was centrifuged. Plasma was collected and the erythrocytes thus obtained were washed three times with an excess of saline. The erythrocytes were hemolyzed by 4 volumes of a hypotonie phosphate buffer (5 mJW, pH 7.0). Selenium and GSH-Px were assayed for the plasma and hemolysate. After the assay of GSH-Px within 2 d, the plasma and hemolysate were frozen at — 20°C and kept until the selenium analysis. Urine samples were taken into plastic tubes. Creatinine, diazoreaction-positive substances (DRPS), and selenium levels were determined. The urine was kept frozen at -20°C until the analyses. The blood and urine sampling was performed from 9 to 11 a.m. on mornings in November and December 1988. Evaluation of Selenium-Rich Food Intake
Since the main selenium sources of the Japanese diet are wheat products, eggs, meat, and fish (Suzuki et al., 1988), dietary selenium intake can be estimated by obtaining information about the intake habit of these foodstuffs. Weekly intake of bread, eggs, meat, and fish was assessed by a simplified questionnaire as described previously (Yoshida, 1990). Assays GSH-Px activity in the plasma and hemolysate was assayed by a modified method of Paglia and Valentine (1967) using 0.29 mW ierf-butyl hydroperoxide as the peroxide substrate (Yoshida et al., 1981). Selenium determination was performed fluorometrically by the method of Watkinson (1966). To ensure the accuracy of selenium determination, NBS bovine liver (NBS-SRM 1577a) was simultaneously measured. MetHb level was measured spectrometrically by the method of Hasegawa et al. (1962). Urinary DRPS was determined by the method of Watanabe et al. (1976) with p-aminophenol as the standard material. Hb and urinary creatinine were measured by the use of commercial kits: Hb, Hemoglobin B-Test
M. YOSHIDA ET AL.
(Wako Pure Chemical Industries Ltd., Osaka); creatinine, Creatinine-Test (Wako). Statistics
Data were evaluated by Student's i-test. The results for the urinary DRPS formed a log-normal distribution, so these data were logarithmically transformed before the statistical analysis.
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RESULTS Levels of Exposure to ANA Compounds
Levels of urinary DRPS and hematological values are shown in Table 1. Urinary DRPS levels of the exposed workers were significantly (p < .001) higher than those of the control group. Because in vivo metabolites of ANA compounds show a positive reaction in the diazo reaction, and urinary DRPS has been utilized as an index of exposure to ANA compounds (Yoshida et al., 1988), the increase of urinary DRPS indicates that the workers in the present study were surely exposed to ANA compounds. In response to the exposure, MetHb levels of the workers were elevated to more than twice those of the control group. Both Ht and Hb values were within the normal ranges in both groups, but were significantly decreased in the exposed workers. Selenium Status
Selenium concentrations and GSH-Px activities in the plasma and erythrocytes are summarized in Table 2. Both plasma and erythrocyte selenium in the exposed workers showed 20% lower values compared to TABLE 1. Urinary Diazo-Reaction-Positive Metabolites and Hematological Values
Urinary diazo metabolites (g p-aminophenol equivalent/ g creatinine)a Hemoglobin (g/100 ml) Hematocrit (%) Methemoglobin (% to Hb)
Control group (n = 107)
Exposed workers (n - 47)
0.33 15.3 45.8 0.5
0.47 (0.20-1.12)b 14.7 ± 0.96 44.5 ± 2.6C 1.2 ± 0.5b
(0.23-0.49) ± 0.9 ± 2.5 ± 0.3
Note. Values except urinary diazo metabolites are means ± SD. Values are geometrical means with SD ranges in the parentheses. ^Significant difference was observed between the control and exposed groups by Student's t-test at p < .001. Significant at p < .05. a
SELENIUM STATUS I N CHEMICAL FACTORY WORKERS
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TABLE 2. Blood and Urinary Selenium Concentrations and Blood Glutathione Peroxidase Activities
Selenium concentration Plasma (ng/ml) Erythrocytes (ng/ml packed cell) Erythrocytes (pg/g Hb) Urine (/¿g/g creatinine) Clutathione peroxidase activity3 Plasma (unit/ml) Erythrocytes (unit/g Hb)
Control group (n " 107)
(n - 47)
118 222 0.67 29
96 177 0.54 27
± ± ± ±
25 60 0.18 12
0.26 ± 0.05 19.0 ± 5.3
Exposed workers
± ± ± ±
166 54b 0.16b 10
0.19 ± 0.046 16.5 ± 3.9C
Note. Values are means ± SD. One unit of glutathione peroxidase activity is expressed as 1 /¿mol of NADPH oxidized per minute. ^Significant difference was observed between the control and exposed groups by Student's f-test a t p < .001. Significant at p < .01. a
those in the control group. Similar to the selenium, the GSH-Px activities were significantly decreased in the exposed workers, but the extent of decrease was different between the plasma and erythrocytes; the plasma GSH-Px was 27% decreased, whereas the erythrocyte enzyme was 13% decreased. In Table 2, urinary selenium excretions are also presented. Unlike the blood selenium, urinary selenium levels in the exposed workers were similar to those in the control group. Habit of Selenium-Rich Food Intake
Table 3 shows weekly intakes of bread, eggs, meat, and fish assessed by the questionnaire. Differences between the exposed workers and the control group were not observed in weekly intakes of the four seleniumTABLE 3. Habits of Bread, Eggs, Meat, and Fish Intake
Bread (slices of white bread/week)a Eggs (number/week) Meat (frequency/week)b Fish (frequency/week)6
Control group (n = 107)
Exposed group (n = 47)
4.8 4.9 5.2 5.1
5.4 5.2 4.7 5.0
± ± ± ±
3.5 2.3 2.5 2.7
± ± ± ±
3.6 2.9 2.8 2.4
Nofe. Values are means + SD. One piece of any other type of bread was regarded as being equivalent to one slice of white bread. frequency of intake at lunch and supper. a
M. YOSHIDAETAL.
rich foods. This indicates a similarity between the two groups in average daily selenium intake.
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DISCUSSION
Generally, the toxic effect of ANA compounds is evaluated by the ability to form MetHb (Watanabe et al., 1976). As a control of the industrial use of MetHb-producing materials such as ANA compounds, the American Conference of Governmental Industrial Hygienists (1988) has proposed a value of 1.5% MetHb as a biological exposure index (BEI), which is a reference value intended as a guideline for the evaluation of potential health hazards, including any chronic effects. In the present study, the workers engaged in the production of dyes or drugs in the chemical factory were surely exposed to ANA compounds. In response to this exposure, the MetHb levels of the workers was apparently increased, and the levels exceeded the BEI in some workers. Duration of the handling of ANA compounds in the factory was 0.7-31.5 (mean ± SD, 14.0 ± 8.7) yr and the amounts of ANA compounds handled remained almost constant over the past two decades; the formation of MetHb at a level around the BEI has therefore continued for a long time in the workers. Long-term methemoglobinemia is considered to result in an anemia; in fact, Hb and Ht values were decreased in the exposed workers. Compared to the control group, blood selenium concentrations and GHS-Px activities in the exposed workers were significantly decreased; selenium status in the exposed workers was therefore apparently lowered. However, careful verification is necessary before the conclusion is drawn that the low selenium status in the workers was associated with occupational exposure to ANA compounds. Dietary selenium intake is the most important factor to contribute to the variation of the selenium status of populations in different geographical areas (Combs and Combs, 1986). As for healthy Japanese populations in a common geographical area, the erythrocyte selenium levels are significantly correlated with the habit of fish intake (Suzuki et al., 1989; Yoshida, 1990). However, as described in Table 3, the intake habits of the four selenium-rich foods including fish were similar for the exposed workers and the controls; average dietary selenium intake is therefore considered to be similar in the two groups. Suzuki et al. (1989) reported that plasma selenium levels were significantly correlated with the selenium intake from wheat products in the diets of subjects prior to blood sampling. On the other hand, we previously observed that urinary selenium excretion before noon was increased in subjects who consumed a large amount of selenium-rich foods at breakfast on the day of the urine sampling and at supper on the preceding day (Yoshida, 1990). In the present study, urinary sele-
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SELENIUM STATUS IN CHEMICAL FACTORY WORKERS
nium levels were similar in the two groups. Although we did not determine the contents of diet before the blood or urine sampling, a similarity in the selenium intake before the sampling between the two groups may exist on the assumption that the rate of selenium excretion is not altered in the exposed workers. Other than the selenium intake, several factors have been known to influence the selenium status in healthy populations; these factors include age (Miller et al., 1983), smoking habits (Ellis et al., 1984), and alcohol abuse (Lloyd et al., 1983). However, as described earlier, differences in these factors did not exist between the exposed and control groups. Accordingly, it is reasonable to consider that the low selenium status in the exposed workers was associated with working at the chemical factory. There have been few studies regarding the selenium status of industrial workers. Lane et al. (1983) reported that selenium concentrations of plasma and erythrocytes and GSH-Px activities of erythrocytes in oil refinery workers in Texas were significantly lower than those in controls in the same region. Similar low selenium status, including low blood and urinary selenium and blood GSH-Px, was also reported in power station and rubber factory workers in Poland (Zachara et al., 1987). In these studies, two explanations for the low selenium status have been proposed: (1) Selenium may be lost in perspiration. (2) A chemical oxidant stress in the occupational environments may influence the distribution and utilization of selenium: This relationship has been suggested on the basis of some animal experiments in which environmental hazards such as polychlorinated biphenyls and organic solvents such as carbon tetrachloride produced tissue peroxidation and exacerbation of selenium deficiency symptoms (Combs and Scott, 1975; Hafeman and Hoekstra, 1977). The present workers must surely have perspired greatly, because the temperature in the working room of the factory was more than 30°C, even in December. Moreover, ANA compounds and their in vivo metabolites have oxidative stress properties such as the formation of MetHb (Beard and Noe, 1981). Thus both proposed explanations seem to be applicable to the present study. However, Levander et al. (1981) pointed out that selenium excretion into sweat occurred only in small amounts, and, unlike the Polish study, the urinary selenium levels were not decreased in the workers. Thus, we consider that only the explanation of a chemical oxidant stress is appropriate for the low selenium status of the exposed workers; that is, the low selenium status was associated with the exposure to ANA compounds. Another possible explanation for the low selenium status in the exposed workers is that the ANA compounds were metabolized to GSH conjugates (Beard and Noe, 1981) and may have affected GSH metabolism (Kosower and Kosower, 1978). In animal experiments, selenium sta-
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M. YOSHIDA ET AL.
tus influences GSH metabolism (Hill and Burk, 1982; Yoshida et al., 1984), and inversely, selenium metabolism is connected with GSH (Hsieh and Ganther, 1971). The low selenium status in the exposed workers may be caused via an altered GSH status. In the present study, not only the selenium levels but also GSH-Px activities were decreased in the exposed workers. According to the New Zealand studies, there is an excellent correlation between selenium levels and GSH-Px activities in whole blood at concentrations below about 0.10 /¿g/ml, or in erythrocytes at concentrations below 0.14 /¿g/ml (Thomson et al., 1977; Rea et al., 1979). Above these concentrations, GSH-Px activity is not correlated with selenium level. Most of the present subjects showed concentrations above these values. Thus, the low GSH-Px activities in the exposed workers may not be directly caused by the low selenium levels. The factor causing the low GSH-Px activities may differ from the factor producing the low selenium levels; there may not be a single cause of the low selenium status. Animal research shows that low selenium status elevates the toxicity of some xenobiotics (Peterson et al., 1982; Combs and Peterson, 1983). Moreover, susceptibility to oxidant stress of erythrocytes, involving hemolysis or MetHb formation, is increased by low selenium status (Rotruck et al., 1972; Kim et al., 1988). Thus, attention must be paid to the low selenium status observed in workers handling ANA compounds. REFERENCES American Conference of Governmental Industrial Hygienists. 1988. Threshold Limit Values and Biological Exposure Indices for 1988-1989, pp. 60-61. Cincinnati: American Conference of Governmental Industrial Hygienists. Beard, R. R., and Noe, J. T. 1981. Aromatic nitro and amino compounds. In Patty's Industrial Hygiene and Toxicology, 3rd rev. ed., eds. G. D. Clayton and F. E. Clayton, vol. 2A, pp. 24132489. New York: Wiley and Sons. Combs, G. F., Jr., and Combs, S. B. 1986. The Role of Selenium in Nutrition, pp. 327-399. New York: Academic Press. Combs, G. F., Jr., and Combs, S. B. 1987. Selenium effects on drug and foreign compound toxicity. Pharmac. Ther. 33:303-315. Combs, G. F., Jr., and Peterson, F. J. 1983. Protection against acute paraquat toxicity by dietary selenium in the chick. J. Nutr. 113:538-545. Combs, G. F., Jr., and Scott, M. L. 1975. Polychlorinated biphenyl-stimulated selenium deficiency in the chick. Poult. Sci. 54:1152-1158. Ellis, N., Lloyd, B., Lloyd, R. S., and Clayton, B. E. 1984. Selenium and vitamin E in relation to risk factors for coronary heart disease. J. Clin. Pathol. 37:200-206. Hafeman, D. G., and Hoekstra, W. G. 1977. Protection against carbon tetrachloride-induced lipid peroxidation in the rat by dietary vitamin E, selenium and measured by ethane evolution. J. Nutr. 107:656-665. Hasegawa, H., Sato, M., Yoshikawa, H., Sakabe, H., Yamaguchi, M., and Hotta, K. 1962. Nitroglycol poisoning in an explosive plant (II). Bull. Natl. Inst. Ind. Health 5:10-20. Hill, K. E., and Burk, R. F. 1982. Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes. J. Biol. Chem. 257:10668-10672.
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Hsieh, H. S., and Canther, H. E. 1971. Acid-volatile selenium formation catalyzed by glutathione reductase. Biochemistry 14:1632-1636. Kim, C-H., Yasumoto, K., Suzuki, T., and Yoshida, M. 1988. Failure of glutathione in suppressing tert-butyl hydroperoxide-induced hemolysis in vitro of selenium-deficient rat erythrocytes. Nutr. Res. 8:767-775. Kok, F. J., Hofman, A., Witteman, J. C. M., de Bruijn, A. M., Kruyssen, D. C. M., de Bruin, M., and Valkenburg, H. A. 1989. Decreased selenium levels in acute myocardial infarction. J. Am. Med. Assoc. 261:1161-1164. Kosower, N. S., and Kosower, E. M. 1978. The glutathione status of cells. Int. Rev. Cytol. 54:109160. Lane, H. W., Warren, D. C., Martin, E., and McCowan, J. 1983. Selenium status of industrial worker. Nutr. Res. 3:805-817. Levander, O. A., Sutherland, B., Morris, V. C., and King, J. C. 1981. Selenium metabolism in human nutrition. In Selenium in Biology and Medicine, 2nd ed., eds. J. E. Spallholz and H. E. Canther, pp. 256-268. Westport, Conn.: AVI. Lloyd, B., Lloyd, R. S., and Clayton, B. E. 1983. Effect of age, smoking, alcohol, and other factors on the selenium status of a healthy population. J. Epidemiol. Community Health 37:213-217. Miller, L., Millis, B. J., Blotcky, A. J., and Lindeman, R. D. 1983. Red blood cell and serum selenium concentrations as influenced by age and selected disease. J. Am. Coll. Nutr. 4:331-341. Paglia, D. E., and Valentine, W. N. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169. Peterson, F. J., Combs, C. F., Jr., Holtzman, J. L., and Mason, R. P. 1982. Effect of selenium and vitamin E deficiency on nitrofurantoin toxicity in the chick. J. Nutr. 112:1741-1746. Rea, H. M., Thomson, C. D., Campbell, D. R., and Robinson, M. F. 1979. Relation between erythrocyte selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities of New Zealand residents and visitors to New Zealand. Br. J. Nutr. 42:201-208. Rotruck, J. T., Pope, A. L., Canther, H. E., and Hoekstra, W. C. 1972. Prevention of oxidative damage to rat erythrocytes by dietary selenium. J. Nutr. 102:689-696. Salonen, J. T., Alfthan, C., Huttunen, J. K., Pikkarainen, J., and Puska, P. 1982. Association between cardiovascular death and myocardial infarction and serum selenium in a matchedpair longitudinal study. Lancet 2:175-179. Salonen, J. T., Alfthan, G., Huttunen, J. K., and Puska, P. 1984. Association between serum selenium and the risk of cancer. Am. J. Epidemiol. 120:342-349. Shamberger, R. J., Tytko, S. A., and Willis, C. E. 1976. Antioxidant and cancer. Part IV. Selenium and age-adjusted cancer mortality. Arch. Environ. Health. 31:231-235. Suzuki, T., Imai, H., Kobayashi, K., Hongo, T., Kashiwazaki, H., Ohtsuka, R., Suzuki, H., and Ishida, H. 1988. Dietary intake of selenium in Japanese. An estimation by analyzed and reported values in foodstuffs and cooked dishes. Nippon Eiyo Shokuryo Cakkaishi (J. Jpn. Soc. Food Nutr. Sci.) 41:91-102. Suzuki, T., Hongo, T., Ohba, T., Kobayashi, K., Imai, H., Ishida, H., and Suzuki, H. 1989. The relation of dietary selenium to erythrocyte and plasma selenium concentrations in Japanese college women. Nutr. Res. 9:839-848. Thomson, C. D., Rea, H. M., Doesburg, V. M., and Robinson, M. F. 1977. Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents. Br. J. Nutr. 37:457-460. Watanabe, T., Ishihara, N., and Ikeda, M. 1976. Toxicity of and biological monitoring for 1,3diamino-2,4,6-trinitrobenzene and other nitro-amino derivatives of benzene and chlorobenzene. Int. Arch. Occup. Environ. Health 37:157-168. Watkinson, J. H. 1966. Fluorometric determination of selenium in biological material with 2,3diaminonaphthalene. Anal. Chem. 38:92-97. Whanger, P. D., Beilstein, M. A., Thomson, C. D., Robinson, M. F., and Howe, M. 1988. Blood selenium and glutathione peroxidase activity of population in New Zealand, Oregon, and South Dakota. FASEB J. 2:2996-3002. Yoshida, M. 1990. Relationship between dietary food consumption assessed by a simplified
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questionnaire and blood or urinary selenium concentration. Nippon Eiyo Shokuryo Gakkaishi (J.Jpn. Soc. Food Nutr. Sci.) 43:49-53. Yoshida, M., Yasumoto, K., Iwami, K., and Tashiro, H. 1981. Distribution of selenium in bovine milk and selenium deficiency in rats fed casein-based diets, monitored by lipid peroxide level and glutathione peroxidase activity. Agric. Biol. Chem. 45:1681-1688. Yoshida, M., Fukunaga, T., Iwami, K., and Yasumoto, K. 1984. Variation of glutathione level and synthesis activity in chick liver due to selenium and vitamin E deficiencies. J. Biochem. 96:1391-1397. Yoshida, M., Sunaga, M., and Hara, I. 1988. Urinary diazo-positive metabolites levels of workers handling p-nitrochIorobenzene in a dye producing factory. Ind. Health 26:87-91. Zachara, B. A., Wasowicz, W., Sklodowska, M., and Gromadzinska, J. 1987. Selenium status, lipid peroxides concentration, and glutathione peroxidase activity in the blood of power station and rubber factory workers. Arch. Environ. Health 42:223-229. Received October 21, 1989 Accepted March 30, 1990
