Sodium arsenite administration via drinking water increases genome-wide and Ha-ras DNA hypomethylation in methyl-deficient C57BL/6J mice.

Arsenic is an established human carcinogen. Deficiencies in available animal models have inhibited a detailed analysis of the mechanism of arsenic induced cancer. This study sought to determine the role of a methyl-deficient diet in combination with sodium arsenite on the genomic methylation status and Ha-ras methylation status of C57BL/6J male mice hepatic DNA. Mice were administered arsenic as sodium arsenite via drinking water at 0, 2.6, 4.3, 9.5 or 14.6 mg sodium arsenite/kg/day. Administration occurred 7 days a week for 130 days. Dose-related effects on the liver were evident in mice administered arsenic and methyl-deficient diets. Most prominent were observations of steatosis and microgranulomas. Sodium arsenite increased genomic hypomethylation in a dose dependent manner and methyl-deficiency and sodium arsenite reduced the frequency of methylation at several cytosine sites within the promoter region of the oncogenic gene, Ha-ras. Methylation changes were prominent in a 500 bp non-CpG island-like region of the Ha-ras promoter and less prominent in a 525 bp CpG island-like region. DNA methylation plays an important role in the physiological expression of many genes including Ha-ras. Significantly reduced methylation at a key regulatory region of Ha-ras in the mouse liver may have relevance to understanding arsenic-induced perturbations in the methylation patterns of cellular growth genes involved in the formation of tumors. These findings highlight the effect of sodium arsenite on inherent methylation processes within the hepatic cell.

Organophosphorous pesticide exposure increases the frequency of sperm sex null aneuploidy.

It has been estimated that 4 of 1,000 live births and 35% of spontaneous abortions are aneuploid and that an important proportion of embryo and newborn aneuploidy is of paternal origin. Exposure to organophosphorous pesticides (OP) has been associated with sperm hyperploidy/polyploidy. Therefore, we aimed to assess the frequency of sperm aneuploidy (X, Y, and 18) and its relationship with urinary OP metabolites in agricultural workers. We performed multicolor fluorescence in situ hybridization on samples from nine men obtained before and during the pesticide spraying season to assess sperm aneuploidy. We measured urinary OP metabolite levels by gas-liquid chromatography. Aneuploidies were found in 0.67% of total sperm nuclei. The most frequent aneuploidy was the lack of a sexual chromosome or sex null (0.19%), followed by XY18 (0.15%) and XY18-18 (0.06%). OP metabolites detected at higher concentrations were dimethylthiophosphate, dimethyldithiophosphate, and diethylphosphate (DEP). There were no differences in average aneuploidy frequency or urinary metabolite levels between samples collected before and after exposure. However, Poisson regression analysis adjusted for age, alcohol intake, and sperm concentration showed significant associations between OP metabolite concentrations and increased frequency of sperm aneuploidies. The association was more evident between DEP and sex null, and the risk increased further during the spraying season. Thus, OP exposure could interfere with sperm chromosome segregation and increase the risk for genetic syndromes, such as Turner's. Further studies are required to assess the prevalence of spontaneous abortions, birth defects, and genetic syndromes in agricultural communities.

Arsenic methylation capacity and skin cancer.

Chronic ingestion of arsenic from drinking water is associated with the occurrence of skin cancer. To clarify the role of arsenic methylation capacity in the development of arsenic-associated skin lesions, an epidemiological case-control study was conducted in the southwestern region of Taiwan, in which 26 skin disorder patients were matched with control subjects. The objective of this study was to determine whether arsenic methylation capacity of patients with skin disorders differed from that of matched controls. Both cases and controls had been exposed to similar high concentrations of arsenic in drinking water. Results indicated that skin lesion cases had higher percents of inorganic arsenic (InAs, 13.1+/-3.7%), methylarsonic acid (MMA, 16.4+/-3.2%), lower percent of dimethylarsinic acid (DMA, 70.5+/-5.8%), and higher ratio of MMA to DMA (MMA/DMA, 0.24+/-0.06) than matched controls (InAs: 11.43+/-2.1%; MMA: 14.6+/-2.6%; DMA: 73.9+/-3.3%; MMA/ DMA: 0.20+/-0.04). Individuals with a higher percentage of MMA (>15.5%) had an odds ratio of developing skin disorder 5.5 times (95% confidence interval, 1.22-24.81) higher than those having a lower percentage of MMA. This association was not confounded by hepatitis B surface antigen, cigarette smoking, or alcohol and tea consumption. It is concluded that arsenic biotransformation including methylation capacity may have a role in the development of arsenic-induced skin disorders.

Approaches to addressing occupational and environmental health needs in Mexico.

The University of California, Los Angeles, has somewhat shifted the focus of its Fogarty program, taking a four-pronged approach: conducting high-level collaborative scientific research with Mexican faculty and trainees at the most advanced institutions in the country; providing training and collaborative research opportunities to faculty/students at other institutions in Mexico (primarily through training faculty who do not hold doctoral degrees); providing environmental and occupational health training to the professional community throughout Mexico; and developing short courses on special topics that provide means for greater research collaboration and skill building. The program is also working with existing institutions to develop academic programs that will enlarge the environmental and occupational health infrastructures in Mexico and Latin America.

Hemoglobin and DNA adduct formation in Fischer-344 rats exposed to 2,4- and 2,6-toluene diamine.

Using gas chromatography/mass spectrometry for detection of hemoglobin adducts, and 32P-postlabelling for DNA adducts, we examined macromolecular binding in Fischer-344 rats administered 2,4-or 2,6-toluene diamine (TDA). The dose-response and correlative relationship between the two macromolecules were investigated over a range of doses (0-250 mg/kg). The time course of adduct formation and removal was also examined. Both TDA isomers induced formation of hemoglobin adducts, but only the 2,4-isomer induced DNA binding. Maximum hemoglobin and DNA adduct levels were detected 24 h following administration. Both hemoglobin and DNA binding increased in a dose-dependent manner. Hemoglobin adduct clearance demonstrated a nonlinear decay, with adduct loss occurring faster than normal erythrocyte clearance. The effects of metabolic inhibitors on adduct formation were examined using piperonyl butoxide and pentachlorophenol to inhibit p450 isozymes and sulfotransferase, respectively. Microsomal enzymatic activation was critical to hemoglobin adduct formation with inhibition by piperonyl butoxide reducing adduct yields by over 90%. Sulfation did not appear to play a significant role in TDA-induced hemoglobin adduct formation.

Determination of hemoglobin adduct levels of the carcinogen 2,4-diaminotoluene using gas chromatography-electron impact positive-ion mass spectrometry.

A procedure to determine hemoglobin adduct yields resulting from exposure to the carcinogen 2,4-diaminotoluene (2,4-TDA) was developed using gas chromatography-electron impact positive-ion mass spectrometry. Liberated 2,4-TDA was quantified following alkaline hydrolysis of hemoglobin. Optimized derivatization of free 2,4-TDA with hepatafluorobutyric anhydride allowed detection of hemoglobin adduct levels as low as 5 ng/g Hb. Pure HFBA-2,4-TDA showed a linear dynamic range of 50 to 5000 pg. The quantitative extraction and recovery of liberated 2,4-TDA (ca. 100%) following hemoglobin hydrolysis allows accurate and precise determinations of adduct yields.

Formation and removal of DNA adducts in Fischer-344 rats exposed to 2,4-diaminotoluene.

32P-Postlabeling was used to examine DNA adduct formation and removal in Fischer-344 rats exposed to the animal carcinogen 2,4-diaminotoluene (DAT). Adduct formation and persistence were compared between target (liver and mammary gland) and non-target organs (kidney and lung) to determine if possible differences could explain the observed organ specificity of DAT induced carcinogenesis. The effects of different exposure conditions on DNA adduct formation and removal were also examined by varying the concentration and frequency of compound administration. DAT produced three distinct DNA adducts. Among the organs examined, DNA binding was highest in the liver, with levels approximately 10 times greater than that of the mammary gland and up to 50 times greater than of the two nontarget sites. Despite the large differences in the initial extent of adduct formation, the persistence of adducts among sites was not significantly different. In the liver, there were dose-dependent differences in DNA adduct formation, but adduct removal following different dosages did not vary significantly. The effects of multiple administration on DNA adduct formation and removal were examined by treating rats with 5 mg/kg DAT daily for 10 consecutive days. Adduct yields from multiple treatment were greater than from a single 50 mg/kg exposure. The persistence of adducts following multiple treatment was also greater than after an equivalent single exposure. The results demonstrated organ-specific and dose-dependent differences in initial extent of DNA adduct formation, but no differences in adduct persistence. However, the results did suggest that adduct formation and persistence may change with repeated administration of DAT.

Aquatic biomonitoring of reclaimed water for potable use: the San Diego Health Effects Study.

Highly treated reclaimed wastewater was evaluated as a possible supplement to raw water sources required to meet San Diego's growing need for potable water. Biomonitoring experiments employing fathead minnows (Pimephales promelas) were used to compare reclaimed water with the city's current raw water supply. Juvenile fish were exposed in flow-through aquaria in field laboratories located at the reclamation plant (AQUA II) and at a municipal potable water treatment facility (Miramar). Biomonitoring measurements were survival and growth, swimming performance, and trace amounts of 68 base/neutral/acid extractable organics, 27 pesticides, and 27 inorganic chemicals found in fish tissues after exposure. Biomonitoring revealed differences in survival, growth, and swimming performance only after 90- and 180-d exposure. Reclaimed water and raw water were not readily distinguishable in 28-d chemical bioaccumulation tests in terms of organic chemical contaminants in fish tissue except for pesticide levels, which tended to be higher in raw water. Similar inorganic species were found in samples from both waters, although there was greater evidence of bioaccumulation of certain contaminants from raw water. Based on biomonitoring parameters included in these experiments, the use of reclaimed water to supplement raw water supplies would appear to pose no major public health threats. The results of these studies will be combined with additional health effects information before final conclusions are reached about the suitability of reclaimed water for human consumption.

Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene.

We used 32P-postlabelling to compare DNA binding between the potent hepatocarcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. The two compounds were compared to determine whether differences in DNA binding could partly explain the differences in their carcinogenicity. Fischer-344 rats were administered 1.2 mmol/kg of a compound by single i.p. injection and examined for DNA adduct formation in the liver. Four adducts were detected following administration of 2,6-dinitrotoluene, with a total adduct yield of 13.5 adducted nucleotides per 10(7) nucleotides. Qualitatively identical adducts were also detected after treatment with the derivative 2-amino-6-nitrotoluene. Adduct yields from 2,6-dinitrotoluene were 30 times greater than from 2-amino-6-nitrotoluene. No adducts were observed following treatment with 2,6-diaminotoluene. 2,6-Dinitrotoluene and 2,6-diaminotoluene were also compared for qualitative differences in hepatotoxicity. 2,6-Dinitrotoluene produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of the latter. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity.

In vitro and in vivo inhibition of lysyl oxidase by aminopropionitriles.

Inhibition of lysyl oxidase (protein-lysine 6-oxidase, EC 1.4.3.13) decreases the rate of collagen and elastin cross-link formation and produces osteolathyrism in animals. Organic nitriles, including beta-aminopropionitrile (BAPN), have been shown to irreversibly inhibit lysyl oxidase in vitro. Both BAPN and 3,3'-iminodipropionitrile (IDPN) have been shown to produce osteolathyric changes when administered to animals. To date compounds that have been reported to inhibit this enzyme possess a primary amine functional group. In this study a series of primary and substituted aminopropionitriles was studied for their ability to inhibit lysyl oxidase activity both in vitro and in vivo. Our results show that of the compounds tested, BAPN was the most potent inhibitor of the enzyme. Reversible inhibition of lysyl oxidase in vitro was found with two secondary aminonitriles, IDPN and monomethylaminopropionitrile (MMAPN). There was no inhibition of enzyme activity associated with the tertiary compound 3,3'-dimethylaminopropionitrile (DMAPN) or propionitrile, a compound lacking an amine functional group. IDPN was found to produce a slight irreversible inhibition of the enzyme both in vitro and in vivo. Pretreatment of rats with pargyline, an inhibitor of monoamine oxidase, was found to increase the inhibitory potential of BAPN (p < or = .1). Pargyline pretreatment did not alter the inhibitory potential for any of the other aminonitriles tested. These results suggest that the presence of a primary amino functional group is not a strict requirement for inhibition of lysyl oxidase. In addition, reversible and irreversible mechanisms of inhibition may be involved in the production of osteolathyric changes associated with IDPN exposure.

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene.

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 mumol/kg. At the highest concentration examined (2046 mumol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10(7) total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 mumol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.

Comparison of DNA adduct formation between 2,4 and 2,6-dinitrotoluene by 32P-postlabelling analysis.

Using 32P-postlabelling, we examined DNA binding by 2,4 and 2,6-dinitrotoluene (DNT) in Fischer-344 rats. DNA binding between the two compounds was compared to determine if differences in adduct formation and persistence could partly explain the known isomer-specific hepatocarcinogenicity of DNTs. The differences in cytotoxicity between the two isomers were also assessed. Both 2,4 and 2,6-DNT induced adduct formation in hepatic DNA. Three distinct adducts were detected following single i.p. administration of 2,4-DNT, while the 2,6-isomer produced four different adducts. Depending on the concentration administered, the two compounds differed in their relative yields. 2,6-DNT produced a greater total adduct yield relative to the 2,4-isomer at low concentrations. Following administration of high concentrations, however, 2,4-DNT predominated. The maximum adduct levels measured were 3.0 and 1.8 adducted nucleotides per 10(6) nucleotides for 2,4 and 2,6-DNT, respectively. Substantial amounts of adducts from both compounds were found to persist over time. After 2 weeks, the mean persistence for 2,4 and 2,6-DNT induced adducts were 42% and 46%, respectively. Qualitative examination for liver toxicity showed 2,6-DNT to be more cytotoxic, inducing extensive hemorrhagic centrilobular necrosis. Rats treated with 2,4-DNT did not show any observable signs of hepatocellular necrosis. Under the conditions of this study, the differences between 2,4 and 2,6-DNT in adduct formation and persistence do not appear to be sufficient to account for their differences in carcinogenicity. The toxicity of 2,6-DNT may be a determining factor in the potent carcinogenicity observed with this compound.

Role of monoamine oxidase in aminopropionitrile-induced neurotoxicity.

Oxidation of aminopropionitriles was measured in vitro with both rat liver mitochondria and bovine plasma monoamine oxidase (MAO). The nonneurotoxic aminonitrile beta-aminopropionitrile (BAPN) was oxidized at a significantly higher rate (p less than .05) than either of the neurotoxic aminonitriles tested; 3,3'-iminodipropionitrile (IDPN) and 3,3'-dimethylaminopropionitrile (DMAPN). DMAPN was a poor substrate for both mitochondrial and plasma MAO. None of the aminonitriles tested were found to inhibit MAO activity in rat brain or liver in vivo. Inhibition of MAO activity with pargyline in vivo did not affect the pattern of IDPN- or DMAPN-induced toxicity. These results suggest that monoamine oxidase is not involved in aminonitrile-induced neurotoxicity.

Characterization of the airborne concentrations of lead in U.S. industry.

Occupational exposure to lead represents a continuing problem of significant magnitude in the United States. To characterize the problem for surveillance purposes, an analysis of the airborne concentrations of lead identified in OSHA compliance inspections was conducted for the years 1979 to 1985. The five specific objectives of the study were: 1) to examine the distribution of air lead concentration in industrial environments; 2) to determine the secular trends in air lead concentrations for high lead industries; 3) to assess which job titles had excessive airborne lead concentrations; 4) to evaluate whether there was a relationship between lead overexposure and company size, unionization, or type of inspection; and 5) to investigate the prevalence of respirator violations for lead. Fifty-two industries were identified which had more than 1/3 of their inspection medians greater than the permissible exposure limit. These included primary and secondary lead smelting, battery manufacture, pigment manufacture, brass/bronze foundries, as well as 46 other industries. There has been little if any improvement in the prevalence and severity of airborne lead concentrations for the high lead industries, battery manufacture, secondary smelting, pigment manufacture, and brass/bronze foundries. Specific high exposure job titles are identified for certain high lead industries. The job title of painting stands out as an especially problematical job title across a number of industries. The prevalence of respirator violations is approximately 20% of all lead inspections.

Workplace smoking policies: attitudes of union members in a high-risk industry.

We surveyed members of a medium-size national union representing workers in high-risk industries to assess workers' support for union and company programs to help smokers break the habit and policies that restrict smoking. Two surveys were conducted that involved 690 respondents in 1984 and 593 respondents in 1985. Respondents overwhelmingly (82%) favored restrictions on smoking in the workplace but less than half agreed that companies or unions should be concerned about workers smoking off the job. For both smokers and nonsmokers, beliefs that cancer has specific causes and can be prevented strongly predict support for workplace smoking control policies. Exposure to company occupational health training also influenced smokers and nonsmokers to support selected smoking control policies. These and other findings led to the conclusion that: (1) educating workers about cancer may promote support for smoking control policies, and (2) smoking control policies are more acceptable in the context of a strong company health and safety program.

Biologic interactions between smoking and occupational exposures.

Cigarette smoking is a major cause of cancer and lung disease in the U.S. population. The biological processes that underlie the response of the lung to cigarette smoke are important considerations for designing analyses of the effects of occupational exposures. Interactions between cigarette smoking and occupational exposures may occur through a combined effect on the mechanism of disease production, through an effect on the dose of the toxic substances that reach the target issue, or through an effect on the response of the lung to the toxic agents. Disease due to occupational exposures can occur in a similar pattern in both smokers and nonsmokers; however, as more complex interactions are examined, different responses to the same occupational exposure may be identified for smokers and nonsmokers. It is only through the successful intermingling of biologic information with epidemiologic data that these interactions can be fully examined.