Blood gene expression signatures predict exposure levels.

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis.

In the previous 500 2-year chemical bioassays within the National Toxicology Program, large intestinal tumors (cecal carcinomas) related to chemical exposure have not been observed in B6C3F1 mice. The recently completed o-nitrotoluene study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. Morphologically, the carcinomas were gland-forming tumors lined by tall columnar epithelial cells that were positive for cytokeratin 20 and negative for cytokeratin 7. Using immunohistochemistry beta-catenin (encoded by Catnb) protein accumulation was detected in 80% (8/10) of the cecal carcinomas, while increased cyclin D1 and p53 protein expression was detected in 73% (8/11), respectively. There was no difference in adenomatous polyposis protein expression between normal colon and cecal carcinomas. All tumors examined exhibited mutations in exon 2 (corresponds to exon 3 in humans) in the Catnb gene. Mutations in p53 were identified in nine of 11 carcinomas, and all were in exon 7. Analysis of the K-ras gene revealed mutations in 82% (9/11) of carcinomas; all had specific G --> T transversions (Gly --> Val) at codons 10 or 12. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations that activate signal transduction pathways (K-ras and Catnb) and changes that disrupt the cell-cycle and bypass G(1) arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene exposure.

Nephrotoxicity and hepatotoxicity induced by inhaled bromodichloromethane in wild-type and p53-heterozygous mice.

Bromodichloromethane (BDCM) is a common municipal drinking water disinfection by-product, resulting in widespread trace human exposure via ingestion and inhalation. The present studies were designed to define organ-specific, BDCM-induced toxicity in wild type (p53(+/+)) and heterozygous (p53(+/-)) mice on both the FVB/N and C57BL/6 genetic backgrounds. Mice were exposed to BDCM vapor daily for 6 h/day and 7 days/week at concentrations of 0, 1, 10, 30, 100, or 150 ppm for 1 week and at 0, 0.3, 1, 3, 10, or 30 ppm for 3 weeks. In the 1-week exposure study, dose-dependent mortality and morbidity were observed at concentrations of 30 ppm and above and were as high as 100% at 150 ppm. In the 3-week exposure study, mortality and morbidity were found only in the 30-ppm exposure groups and were 0, 17, 67, and 33% for the wild-type C57BL/6, p53(+/-) C57BL/6, wild-type FVB/N, and p53(+/-) FVB/N mice, respectively. BDCM was a particularly potent kidney cytotoxicant. Dose-dependent tubular degeneration, necrosis, and associated regenerative cell proliferation greater than 10-fold over controls were seen at concentrations as low as 10 ppm in the kidneys of all strains at 1 week. Similar dose-dependent increases in hepatic necrosis, degeneration, and regenerative cell proliferation were observed but were induced only at concentrations of 30 ppm and higher. Pathological changes were more severe in the FVB/N compared to the C57BL/6 mice and were more severe in the heterozygotes compared to the wild-type mice. However, recovery and return of the percentage of kidney cells in S-phase to control levels was seen at 3 weeks. The estimated maximum tolerated dose for longer-term exposures was 15 ppm, based on mortality, induced kidney pathology, and regenerative cell proliferation. A one-year cancer bioassay was initiated with doses of 0, 0.5, 3, 10, and 15 ppm, based on this information. No pathological changes in the livers were found at the 13-week time point of that study. At 13 weeks, the kidney lesions and regenerative cell proliferation seen at the 1-week time point at doses of 10 ppm and above had resolved, and the cell proliferation rates had returned to baseline. Differences in toxicity indicate that caution be used in substituting wild-type mice for transgenic mice for range-finding studies to select doses for p53(+/-) cancer studies. Resolution of the kidney lesions indicates that periods of very high regenerative cell proliferation, potentially important in the carcinogenic process, may not be observed if measurements are taken only at 3 weeks of exposure or later.

Subchronic sodium chlorate exposure in drinking water results in a concentration-dependent increase in rat thyroid follicular cell hyperplasia.

Chlorine dioxide (ClO2) is an effective drinking water disinfectant, but sodium chlorate (NaClO3) has been identified as a potentially harmful disinfection by-product. Studies were performed to describe the development of thyroid lesions in animals exposed to NaClO3 in the drinking water. Male and female F344 rats and B6C3F1 mice were exposed to 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/L NaClO3 for 21 days. Additional male F344 rats were exposed to 0, 0.001. 0.01. 0.1, 1.0. or 2.0 g/L NaClO3 for 90 days. Female F344 rats were exposed to 0, 0.5, 1.0, 2.0, 4.0, or 6.0 g/L of NaClO3 for 105 days. Thyroid tissues were processed by routine methods for light microscopic examination, and follicular cell hyperplasia was diagnosed using a novel method. Thyroid hormone levels were altered significantly after 4 and 21 days. NaClO, treatment induced a concentration-dependent increase in the incidence and severity of thyroid follicular cell hyperplasia. Male rats are more sensitive to the effects of NaClO3 treatment than females. Follicular cell hyperplasia was not present in male or female B6C3F1 mice. These data can be used to estimate the human health risk that would be associated with using ClO2, rather than chlorine, to disinfect drinking water.

Point mutations of K-ras and H-ras genes in forestomach neoplasms from control B6C3F1 mice and following exposure to 1,3-butadiene, isoprene or chloroprene for up to 2-years.

1,3 Butadiene (BD), isoprene (IP) and chloroprene (CP) are structural analogs. There were significantly increased incidences of forestomach neoplasms in B6C3F1 mice exposed to BD, IP or CP by inhalation for up to 2-years. The present study was designed to characterize genetic alterations in K- and H-ras proto-oncogenes in a total of 52 spontaneous and chemically induced forestomach neoplasms. ras mutations were identified by restriction fragment length polymorphism, single strand conformational polymorphism analysis, and cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded forestomach neoplasms. A higher frequency of K- and H-ras mutations was identified in BD-, IP- and CP-induced forestomach neoplasms (83, 70 and 57%, respectively, or combined 31/41, 76%) when compared to spontaneous forestomach neoplasms (4/11, 36%). Also a high frequency of H-ras codon 61 CAA-->CTA transversions (10/41, 24%) was detected in chemically induced forestomach neoplasms, but none were present in the spontaneous forestomach neoplasms examined. Furthermore, an increased frequency (treated 13/41, 32% versus untreated 1/11, 9%) of GGC-->CGC transversion at K-ras codon 13 was seen in BD-, and IP-induced forestomach neoplasms, similar to the predominant K-ras mutation pattern observed in BD-induced mouse lung neoplasms. These data suggest that the epoxide intermediates of the structurally related chemicals (BD, IP, and CP) may cause DNA damage in K-ras and H-ras proto-oncogenes of B6C3F1 mice following inhalation exposure and that mutational activation of these genes may be critical events in the pathogenesis of forestomach neoplasms induced in the B6C3F1 mouse.

The effect of short intermittent light exposures on the melatonin circadian rhythm and NMU-induced breast cancer in female F344/N rats.

We investigated the effects of altered endogenous nighttime melatonin concentrations on mammary tumor production in an N-nitroso-N-methylurea (NMU)-induced breast cancer model in female Fischer 344 (F344)/N rats. Experiments were designed 1) to evaluate whether short-duration intermittent exposures to light at night would affect the nocturnal rise of melatonin, resulting in a decrease in nighttime serum melatonin concentrations, 2) to evaluate whether any suppression of nighttime serum melatonin concentrations could be maintained for a period of weeks, and 3) to determine the effects of suppressed serum melatonin concentrations on the incidence and progression of NMU-induced breast cancer. In vivo studies were used to assess serum melatonin concentrations after 1 day and 2 and 10 weeks of nightly administration of short-duration intermittent light exposure at night and incidence of NMU-induced tumors. Five 1-minute exposures to incandescent light every 2 hours after the start of the dark phase of the light: dark cycle decreased the magnitude of the nocturnal rise of serum melatonin concentrations in rats by approximately 65%. After 2 weeks of nightly intermittent light exposures, an average decrease of the peak nighttime serum melatonin concentrations of approximately 35% occurred. The amelioration continued and, at 10 weeks, peak nighttime serum melatonin concentrations were still decreased, by approximately 25%. Because peak endogenous nighttime serum melatonin values could be moderately suppressed for at least 10 weeks, a 26-week NMU mammary tumor study was conducted. Serum melatonin concentrations and incidence, multiplicity, and weight of NMU-induced mammary tumors were assessed. A group of pinealectomized (Px) animals was also included in the tumor study. No effect on the development of mammary tumors in an NMU-induced tumor model in rats occurred when endogenous nighttime serum melatonin concentrations were moderately suppressed by short-duration intermittent light exposures at night. At necropsy, there were no alterations in mammary tumor incidence (28/40 NMU controls, 28/40 NMU + light, 31/40 NMU + Px), multiplicity (2.18 tumors/tumor-bearing NMU control, 1.89 NMU + light, 2.39 NMU + Px), or average tumor weight (1.20 g NMU control, 1.19 g NMU + light, 0.74 g NMU + Px). Tumor burden had no effect on the serum melatonin cycle. At 26 weeks, however, animals exposed to intermittent light at night exhibited approximately 3-fold higher serum melatonin concentrations as compared with controls. Additionally, rats that had been pinealectomized at 4 weeks of age had serum melatonin concentrations that were markedly higher than the expected baseline concentrations for pinealectomized rats (<15 pg/ml), suggesting the reestablishment of a melatonin cycle. This finding was unexpected and suggests that melatonin can be produced by an organ or tissue other than the pineal gland.

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years.

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.

Magnetic fields and mammary cancer in rodents: a critical review and evaluation of published literature.

Epidemiological data suggesting a possible increase in breast cancer risk in male electricians have raised concerns about the relationship between exposure to power-frequency magnetic fields and breast cancer. In this paper, we review the results of animal studies that are relevant to identifying possible increases in breast cancer risk resulting from exposure to 50 or 60 Hz magnetic fields. Three large-scale chronic bioassays of carcinogenesis in rats or mice exposed to magnetic fields for 2 years demonstrated no increases in the incidence of mammary cancer; it is generally accepted that power-frequency magnetic fields have little or no activity as a complete carcinogen in the rodent mammary gland. Findings from one laboratory, though inconsistent, suggest that magnetic fields may stimulate mammary neoplasia in rats treated with a chemical carcinogen. However, studies conducted in two other laboratories failed to confirm these findings; rats exposed to magnetic fields demonstrated patterns of tumor incidence, multiplicity, size and latency that were generally similar to those in sham-exposed controls. Where differences were seen, the groups exposed to magnetic fields generally had fewer mammary tumors than did sham-exposed controls. On this basis, evaluations of the activity of 50 or 60 Hz magnetic fields in models of multistage mammary cancer in rodents have generally been negative; positive findings have been reported from only one laboratory. The totality of rodent data does not support the hypothesis that power-frequency magnetic-field exposure enhances mammary cancer in rodents, nor does it provide experimental support for possible epidemiological associations between magnetic-field exposure and increased breast cancer risk.

Leukemia and lymphoma incidence in rodents exposed to low-frequency magnetic fields.

A weak association between residential or occupational exposure to electric and magnetic fields (50/60 Hz fields) and an increased incidence of leukemia has been reported. Numerous animal studies have evaluated the potential association between magnetic-field exposure and leukemia. These include long-term (up to 2(1/2) years) bioassays, initiation/promotion studies, investigations in transgenic models, and tumor growth studies. Exposure to 60 Hz circularly polarized magnetic fields at 1,400 microT for 28 months did not affect lymphoma incidence in mice. The study included over 2000 C57BL/6J mice. In another study, 1000 B6C3F(1) mice exposed to 60 Hz magnetic fields up to 1000 microT for 2 years showed no increase in lymphomas. Approximately 400 transgenic Emu-Pim1 mice exposed to 50 Hz fields up to 1000 microT for up to 18 months had no increased incidence of leukemia. Similarly, Trp53(+/-) mice and Pim1transgenic mice exposed to 60 Hz magnetic fields for 23 weeks showed no increased incidence of lymphoma. Three studies in F344 rats exposed to 50 or 60 Hz magnetic fields up to 5 mT showed no increased incidence of leukemia. The combined animal bioassay results are nearly uniformly negative for magnetic-field exposures enhancing leukemia and weaken the possible epidemiological association between magnetic-field exposures and leukemia in humans as suggested by epidemiological data.

Evaluation of in vitro effects of 50 and 60 Hz magnetic fields in regional EMF exposure facilities.

A weak association between magnetic-field exposure and increased incidences of cancer has been reported. While alterations in cellular processes after in vitro magnetic-field exposures have also been reported to provide plausibility for this association, other laboratories have been unable to repeat the findings. As part of an accelerated electric- and magnetic-field (EMF) research program, the National Institute of Environmental Health Sciences with the Department of Energy identified the replication of the published positive effects as a priority. Regional EMF exposure facilities were established to investigate major in vitro effects from the literature. These included effects on gene expression, intracellular calcium, colony growth in soft agar, and ornithine decarboxylase activity. The laboratories that first reported these effects provided experimental protocols, cell lines, and other relevant experiment details. Regional facility studies included sham/sham exposures (no applied field in either chamber) and were done in a blinded fashion to minimize investigator bias. In nearly all experiments, no effects of magnetic-field exposure were found. The effort provided insight into dealing with the difficulty of replication of subtle effects in complex biological systems. Experimental techniques provided some clues for the differences in experimental results between the regional facility and the original investigator. Studies of subtle effects require extraordinary efforts to confirm that the effect can be attributed to the applied exposure.

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Re-evaluation of the 2-year chloroform drinking water carcinogenicity bioassay in Osborne-Mendel rats supports chronic renal tubule injury as the mode of action underlying the renal tumor response.

Chloroform, generally regarded as a non-genotoxic compound, is associated with the induction of liver and/or kidney tumors in laboratory mice and rats. In particular, chloroform produced renal tubule tumors in low incidence in male Osborne-Mendel rats when administered by corn-oil gavage or in the drinking water. There is a lack of data on intermediate endpoints that may be linked to renal cancer development in this strain of rat, in contrast to mice. Specifically, evidence linking chloroform-induced liver and kidney tumors in mice with cytotoxicity and regenerative cell proliferation is very strong, but weak in the rat. In the present study, kidney tissue from a carcinogenicity bioassay of chloroform in Osborne-Mendel rats was re-evaluated for histological evidence of compound-induced cytotoxicity and cell turnover. All rats treated with 1800 ppm (160 mg/kg/day, high-dose group) in the drinking water for 2 years and half the rats treated with 900 ppm (81 mg/kg/day) had mild to moderate changes in proximal convoluted tubules in the mid to deep cortex indicative of chronic cytotoxicity. Tubule alterations specifically associated with chronic chloroform exposure included cytoplasmic basophilia, cytoplasmic vacuolation, and nuclear crowding consistent with simple tubule hyperplasia. Occasional pyknotic cells, mitotic figures in proximal tubules, and prominent karyomegaly of the renal tubule epithelium were present. These alterations were not present in control groups or at the 200-ppm (19 mg/kg/day) or 400-ppm (38 mg/kg/day) dose levels. This new information adds substantially to the weight of evidence that the key events in chloroform-induced carcinogenicity in rat kidney include sustained cellular toxicity and chronic regenerative hyperplasia.

Examination of low-incidence brain tumor responses in F344 rats following chemical exposures in National Toxicology Program carcinogenicity studies.

Neoplasms in the brain are uncommon in control Fischer 344 (F344) rats; they occur at a rate of less than 1% in 2-yr toxicity/carcinogenicity studies. Furthermore, only 10 of nearly 500 studies conducted by the National Toxicology Program (NTP) showed any evidence of chemically related neoplastic effects in the brain. Generally, the brain tumor responses were considered equivocal, because the characteristics of potential neurocarcinogenic agents (such as statistically significant increased incidences, decreased latency and/or survival, and demonstration of dose-response relationships) were not observed. A thorough examination, including comparisons with a well-established historical database, is often critical in evaluating rare brain tumors. Chemicals that gave equivocal evidence of brain tumor responses were generally associated with carcinogenicity at other sites, and many chemicals were mutagenic when incubated with metabolic activating enzymes. Other factors that were supportive of the theory that marginal increases in brain tumor incidence were related to chemical exposure were that (a) some of the tumors were malignant, (b) no brain neoplasms were observed in concurrent controls from some studies, and/or (c) brain tumors were also seen following exposure to structurally related chemicals. In 2-yr studies in F344 rats (studies conducted by the NTP), equivocal evidence of carcinogenicity was observed for the following 9 chemicals: isoprene, bromoethane, chloroethane, 3,3'-dimethylbenzidine dihydrochloride, 3,3'-dimethoxybenzidine dihydrochloride, furosemide, C.I. direct blue 15, diphenhydramine hydrochloride, and 1-H-benzotriazole. Glycidol was the only chemical evaluated by the NTP with which there was clear evidence of brain tumor induction in F344 rats. Clarification of the potential neurocarcinogenic risks of chemicals that produce equivocal evidence of a brain tumor response in conventional 2-yr rodent studies may be aided by the use of transgenic mouse models that exhibit genetic alterations that reflect those present in human brain tumors as well as by the use of in utero exposures.

Effect of 13 week magnetic field exposures on DMBA-initiated mammary gland carcinomas in female Sprague-Dawley rats.

Several studies suggest that exposure to 50 Hz magnetic fields may promote chemically induced breast cancer in rats. Groups of 100 female Sprague-Dawley rats were initiated with four weekly 5 mg gavage doses of 7,12-dimethylbenz[a]anthracene (DMBA) starting at 50 days of age. After the first weekly DMBA administration, exposure to ambient fields (sham exposed), 50 Hz magnetic fields at either 1 or 5 G field intensity or 60 Hz fields at 1 G for 18.5 h/day, 7 days/week was initiated. Exposure continued for 13 weeks. A vehicle control group without DMBA was included. In a second study, using lower doses of DMBA, groups of 100 female Sprague-Dawley rats were initiated with four weekly doses of 2 mg of DMBA starting at 50 days of age followed, after the first weekly DMBA administration, by exposure to ambient fields (sham exposed) or 50 Hz magnetic fields at either 1 or 5 G field intensity for 18.5 h/day, 7 days/week for 13 weeks. Rats were weighed and palpated weekly for the presence of tumors. There was no effect of magnetic field exposure on body weight gains or on the time of appearance of mammary tumors in either study. At the end of 13 weeks, the animals were killed and the mammary tumors counted and measured. Mammary gland masses found grossly were examined histologically. In the first 13 week study, the mammary gland carcinoma incidences were 92, 86, 96 and 96% for the DMBA controls, 1 G, 50 Hz, 5 G, 50 Hz and 1 G, 60 Hz groups, respectively. The total numbers of carcinomas were 691, 528 (P < 0. 05, decrease), 561 and 692 for the DMBA controls, 1 G, 50 Hz, 5 G, 50 Hz and 1 G, 60 Hz groups, respectively. In study 2, the mammary gland carcinoma incidences were 43, 48 and 38% for the DMBA controls, 1 G, 50 Hz and 5 G, 50 Hz groups, respectively. The total numbers of carcinomas were 102, 90 and 79 for the DMBA controls, 1 G, 50 Hz and 5 G, 50 Hz groups, respectively. There was no effect of magnetic field exposure on tumor size either by in-life palpation or by measurement at necropsy in either study. There was no evidence that 50 or 60 Hz magnetic fields promoted breast cancer in these studies in female rats. These studies do not support the hypothesis that magnetic field exposure promotes breast cancer in this DMBA rat model.

Chronic toxicity/oncogenicity evaluation of 60 Hz (power frequency) magnetic fields in F344/N rats.

A 2-yr whole-body exposure study was conducted to evaluate the chronic toxicity and possible oncogenicity of 60 Hz (power frequency) magnetic fields in rats. Groups of 100 male and 100 female F344/N rats were exposed continuously to pure, linearly polarized, transient-free 60 Hz magnetic fields at flux densities of 0 Gauss (G) (sham control), 20 milligauss (mG), 2 G, and 10 G; an additional group of 100 male and 100 female F344/N rats received intermittent (1 hr on/1 hr off) exposure to 10 G fields. Mortality patterns, body weight gains throughout the study, and the total incidence and number of malignant and benign tumors in all groups exposed to magnetic fields were similar to those found in sex-matched sham controls. Statistically significant increases in the combined incidence of C-cell adenomas and carcinomas of the thyroid were seen in male rats chronically exposed to 20 mG and 2 G magnetic fields. These increases were not seen in male rats exposed continuously or intermittently to 10 G fields or in female rats at any magnetic field exposure level. No increases in the incidence of neoplasms, which have been identified in epidemiology studies as possible targets of magnetic field action (leukemia, breast cancer, and brain cancer), were found in any group exposed to magnetic fields. There was a decrease in leukemia in male rats exposed to 10 G intermittent fields. The occurrence of C-cell tumors at the 2 lower field intensities in male rats is interpreted as equivocal evidence of carcinogenicity; data from female rats provides no evidence of carcinogenicity in that sex. These data, when considered as a whole, are interpreted as indicating that chronic exposure to pure linearly polarized 60 Hz magnetic fields has little or no effect on cancer development in the F344/N rat.

Chronic toxicity/oncogenicity evaluation of 60 Hz (power frequency) magnetic fields in B6C3F1 mice.

A 2-yr whole-body exposure study was conducted to evaluate the chronic toxicity and possible oncogenicity of 60 Hz (power frequency) magnetic fields in mice. Groups of 100 male and 100 female B6C3F1 mice were exposed to pure, linearly polarized, transient-free 60 Hz magnetic fields at flux densities of 0 Gauss (G) (sham control), 20 milligauss (mG), 2 G, and 10 G; an additional group of 100 male and 100 female B6C3F1 mice received intermittent (1 hr on/1 hr off) exposure to 10 G fields. A small but statistically significant increase in mortality was observed in male mice exposed continuously to 10 G fields; mortality patterns in all other groups of mice exposed to magnetic fields were comparable to those found in sex-matched sham controls. Body weight gains and the total incidence and number of malignant and benign tumors were similar in all groups. Magnetic field exposure did not increase the incidence of neoplasia in any organ, including those sites (leukemia, breast cancer, and brain cancer) that have been identified in epidemiology studies as possible targets of magnetic field action. A statistically significant decrease in the incidence of malignant lymphoma was observed in female mice exposed continuously to 10 G fields, and statistically significant decreases in the incidence of lung tumors were seen in both sexes exposed continuously to 2 G fields. These data do not support the hypothesis that chronic exposure to pure, linearly polarized 60 Hz magnetic fields is a significant risk factor for neoplastic development in mice.

Mutations in ras genes in experimental tumours of rodents.

Studies of carcinogenesis in rodents are valuable for examining mutagenesis in vivo. An advantage of evaluating the frequency and spectra of ras mutations in chemically induced neoplasms is that the additional data at the molecular level indicate whether the carcinogenic effect is due to the chemical and is not a spontaneous event, as illustrated by the numerous examples in Appendices 1 and 2. In addition, data on the frequency and spectra of ras mutations in spontaneous and chemically induced neoplasms clearly expand the toxicological database by providing information helpful for understanding the pathogenesis of carcinogenesis. For example: (1) ozone-induced lung neoplasms had two unique mutations, one (codon 61 K-ras CTA mutation) consistent with a direct genotoxic event and a second (codon 12 K-ras G --> T transversion) consistent with an indirect genotoxic effect; (2) isoprene-induced Harderian gland neoplasms had a unique K-ras A --> T transversion at codon 61 which provided evidence that formation of an epoxide intermediate was involved; (3) 1,3-butadiene-induced neoplasms had a characteristic K-ras G --> C transversion mutation at codon 13 which was also consistent with a chemical-specific effect; (4) methylene chloride-induced liver neoplasms had an H-ras mutation profile at codon 61 similar to that of spontaneous tumours, suggesting that methylene chloride promotes cells with 'spontaneously initiated' ras mutations and (5) oxazepam-induced liver neoplasms had a low frequency of ras mutations, suggesting a nonmutagenic pathway of carcinogenesis. By extending the evaluation of rodent tumours to include molecular studies on ras mutation spectra and abnormalities in other cancer genes with human homologues, a number of hypotheses can be tested, allowing the most complete understanding of carcinogenesis in rodents and in potential extrapolation to the human risk situation.

Drinking water disinfection byproducts: review and approach to toxicity evaluation.

There is widespread potential for human exposure to disinfection byproducts (DBPs) in drinking water because everyone drinks, bathes, cooks, and cleans with water. The need for clean and safe water led the U.S. Congress to pass the Safe Drinking Water Act more than 20 years ago in 1974. In 1976, chloroform, a trihalomethane (THM) and a principal DBP, was shown to be carcinogenic in rodents. This prompted the U.S. Environmental Protection Agency (U.S. EPA) in 1979 to develop a drinking water rule that would provide guidance on the levels of THMs allowed in drinking water. Further concern was raised by epidemiology studies suggesting a weak association between the consumption of chlorinated drinking water and the occurrence of bladder, colon, and rectal cancer. In 1992 the U.S. EPA initiated a negotiated rulemaking to evaluate the need for additional controls for microbial pathogens and DBPs. The goal was to develop an approach that would reduce the level of exposure from disinfectants and DBPs without undermining the control of microbial pathogens. The product of these deliberations was a proposed stage 1 DBP rule. It was agreed that additional information was necessary on how to optimize the use of disinfectants while maintaining control of pathogens before further controls to reduce exposure beyond stage 1 were warranted. In response to this need, the U.S. EPA developed a 5-year research plan to support the development of the longer term rules to control microbial pathogens and DBPs. A considerable body of toxicologic data has been developed on DBPs that occur in the drinking water, but the main emphasis has been on THMs. Given the complexity of the problem and the need for additional data to support the drinking water DBP rules, the U.S. EPA, the National Institute of Environmental Health Sciences, and the U.S. Army are working together to develop a comprehensive biologic and mechanistic DBP database. Selected DBPs will be tested using 2-year toxicity and carcinogenicity studies in standard rodent models; transgenic mouse models and small fish models; in vitro mechanistic and toxicokinetic studies; and reproductive, immunotoxicity, and developmental studies. The goal is to create a toxicity database that reflects a wide range of DBPs resulting from different disinfection practices. This paper describes the approach developed by these agencies to provide the information needed to make scientifically based regulatory decisions.

High frequency of codon 61 K-ras A-->T transversions in lung and Harderian gland neoplasms of B6C3F1 mice exposed to chloroprene (2-chloro-1,3-butadiene) for 2 years, and comparisons with the structurally related chemicals isoprene and 1,3-butadiene.

Chloroprene is the 2-chloro analog of 1,3-butadiene, a potent carcinogen in laboratory animals. Following 2 years of inhalation exposure to 12.8, 32 or 80 p.p.m. chloroprene, increased incidences of lung and Harderian gland (HG) neoplasms were observed in B6C3F1 mice at all exposure concentrations. The present study was designed to characterize genetic alterations in the K- and H-ras proto-oncogenes in chloroprene-induced lung and HG neoplasms. K-ras mutations were detected in 80% of chloroprene-induced lung neoplasms (37/46) compared with only 30% in spontaneous lung neoplasms (25/82). Both K- and H-ras codon 61 A-->T transversions were identified in 100% of HG neoplasms (27/27) compared with a frequency of 56% (15/27) in spontaneous HG neoplasms. The predominant mutation in chloroprene-induced lung and HG neoplasms was an A-->T transversion at K-ras codon 61. This mutation has not been detected in spontaneous lung tumors of B6C3F1 mice and was identified in only 7% of spontaneous HG neoplasms. In lung neoplasms, greater percentages (80 and 71%) of A-->T transversions were observed at the lower exposures (12.8 and 32 p.p.m.), respectively, compared with 18% at the high exposure. In HG neoplasms, the percentage of A-->T transversions was the same at all exposure concentrations. The chloroprene-induced ras mutation spectra was similar to that seen with isoprene, where the predominant base change was an A-->T transversion at K-ras codon 61. This differed from 1,3-butadiene, where K-ras codon 13 G-->C transitions and H-ras codon 61 A-->G transitions were the predominant mutations. The major finding of K-ras A-->T transversions in lung and Harderian gland neoplasms suggests that this mutation may be important for tumor induction by this class of carcinogens.

Effect of 26 week magnetic field exposures in a DMBA initiation-promotion mammary gland model in Sprague-Dawley rats.

Several studies have suggested that exposure to 50 Hz magnetic fields promote chemically induced breast cancer in rats. Groups of 100 female Sprague-Dawley rats were initiated with a single 10 mg gavage dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age followed by exposure to ambient fields (sham exposed), 50 Hz magnetic fields at either 1 or 5 Gauss (G) field intensity or 60 Hz fields at 1 G for 18.5 h/day, 7 days/week for 26 weeks. A vehicle control group without DMBA was included. Rats were palpated weekly for the presence of tumors. There was no effect of magnetic field exposure on body weight gains or the time of appearance of mammary tumors. At the end of 26 weeks, the animals were killed and the mammary tumors counted and measured. Mammary gland masses found grossly were examined histologically. The mammary gland carcinoma incidence was 96, 90, 95 and 85% (P < 0.05, decrease) for the DMBA controls, 1 G 50 Hz, 5 G 50 Hz and 1 G 60 Hz groups, respectively. The total numbers of carcinomas were 649, 494 (P < 0.05, decrease), 547 and 433 (P < 0.05, decrease) for the DMBA controls, 1 G 50 Hz, 5 G 50 Hz and 1 G 60 Hz groups, respectively. The number of fibroadenomas varied from 276 to 319, with the lowest number in the 1 G 60 Hz exposure group. Measurement of the tumors revealed no difference in tumor size between groups. In this breast cancer initiation-promotion study in female Sprague-Dawley rats, there was no evidence that 50 or 60 Hz magnetic fields promoted breast cancer under the conditions of this assay. This study does not support the hypothesis that magnetic field exposure can promote breast cancer in this rat model.