F. Bernard Daniel

The Carcinogenicity of Dichloroacetic Acid in the Male B6C3F1 Mouse

Groups of male B6C3F1 mice (N = 50) were provided drinking water containing 2 g/liter sodium chloride (control) and 0.05, 0.5, and 5 g/liter dichloroacetic acid (DCA). Treatment of 30 animals in each group was carried out to 60 or 75 weeks. In a separate experiment, mice exposed to 3.5 g/liter DCA and the corresponding acetic acid control group were killed at 60 weeks. Groups of 5 mice were killed at 4, 15, 30, and 45 weeks. Time-weighted mean daily doses of 7.6, 77, 410, and 486 mg/kg/day were calculated for 0.05, 0.5, 3.5, and 5 g/liter DCA treatments. Animals exposed to 3.5 and 5 g/liter DCA had final body weights that were 87 and 83%, respectively, of the control value. Relative liver weights of 136, 230, and 351% of the control value were measured for 0.5, 3.5, and 5 g/liter, respectively. At 60 weeks mice receiving 5.0 g/liter DCA had a 90% prevalence of liver neoplasia with a mean multiplicity of 4.50 tumors/animal. Exposure to 3.5 g/liter DCA for 60 weeks resulted in a 100% tumor prevalence with an average of 4.0 tumors/animal. The prevalence of liver neoplasia and tumor multiplicity at 60 and 75 weeks in the 0.05 g/liter DCA (24.1%; 0.31 tumors/animal) and in the 0.5 g/liter group (11.1%; 0.11 tumors/animal) did not differ significantly from the control value (7.1% and 0.07 tumors/animal). No liver tumors were found in the group treated with acetic acid. Hyperplastic nodules were seen in the 3.5 (58%; 0.92/animal) and 5 g/liter DCA groups (83%; 1.27/animal). There was a significant positive dose-related trend in the age-adjusted prevalence of liver tumors. These data confirm the hepatocarcinogenicity of DCA administered in the drinking water to male B6C3F1 mice for 60 weeks. The results together with those in an earlier report from this laboratory suggest, for the conditions under which these assays were conducted, a threshold concentration of at least 0.5 g/liter followed by a steep rise to a maximum tumor incidence at 2 g/liter DCA.

Hepatocarcinogenicity of chloral hydrate, 2-chloroacetaldehyde, and dichloroacetic acid in the male B6C3F1 mouse☆

The chlorinated acetaldehydes, chloral hydrate (CH) and 2-chloroacetaldehyde (CAA), have been identified as chlorination by-products in finished drinking water supplies. Although both chemicals are genotoxic, their potential for carcinogenicity had not been adequately explored. The studies reported here are chronic bioassays conducted with male B6C3F1 mice exposed to levels of 1 g/liter CH and 0.1 g/liter CAA via the drinking water for 104 weeks. Distilled water (H2O) served as the untreated control and dichloroacetic acid (DCA; 0.5 g/liter), another chlorine disinfection by-product, was included. The mean daily ingested doses were approximately 166 mg/kg/day for CH, 17 mg/kg/day for CAA, and 93 mg/kg/day for DCA. Evaluations included mortality, body weight, organ weights, gross pathology, and histopathology. The primary target organ was the liver as the organ weights and pathological changes in the other organs (spleen, kidneys, and testes) were comparable between the treated groups and the H2O control group. Liver weights were increased for all three test chemicals at the terminal euthanasia with the greatest increase seen in the CH and DCA groups. Hepatocellular necrosis was induced by all three test chemicals, and it was also most prevalent and severe in the CH and DCA groups. A significant increase in the prevalence of liver tumors was seen for all three chemicals. The strongest response was with DCA, in which 63% of the 104-week survivors had hepatocellular carcinomas (carcinomas) and 42% possessed hepatocellular adenomas (adenomas) and the combined prevalence for carcinomas plus adenoma was 75%. The corresponding prevalence rate for carcinomas, adenomas, and combined tumors were 46, 29, and 71%; 31, 8, and 38%; and 10, 5, and 15% for CH, CAA, and H2O, respectively. In addition to the tumors we evaluated the prevalence of a possible preneoplastic lesion, the hepatocellular hyperplastic nodule (nodules), a lesion which occurred in all three treated groups but not in the H2O group.

Tissue distribution, excretion, and urinary metabolites of dichloroacetic acid in the male fischer 344 rat

The disposition of dichloroacetic acid (DCA) was investigated in Fischer 344 rats over the 48 h after oral gavage of 282 mg/kg of 1- or 2-[14C]-DCA (1-DCA or 2-DCA) and 28.2 mg/kg of 2-DCA. DCA was absorbed quickly, and the major route of disposition was through exhalation of carbon dioxide and elimination in the urine. The dispositions of 1- and 2-DCA at 282 mg/kg were similar. With 2-DCA, the disposition differed with dose in that the percentage of the dose expired as carbon dioxide decreased from 34.4% (28.2 mg/kg) to 25.0% (282 mg/kg), while the percentage of the radioactivity excreted in the urine increased from 12.7 to 35.2%. This percentage increase in the urinary excretion was mostly attributable to the presence of unmetabolized DCA, which comprised more than 20% at the higher dose and less than 1% at the lower dose. The major urinary metabolites were glycolic acid, glyoxylic acid, and oxalic acid. DCA and its metabolites accumulated in the tissues and were eliminated slowly. After 48 h, 36.4%, 26.2%, and 20.8% of the dose was retained in the tissues of rats administered 28.2 and 282 mg/kg of 2-DCA and 282 mg/kg of 1-DCA, respectively. Of the organs examined, the liver (4.9-7.9% of dose) and muscle (4.5-9.9%) contained the most radioactivity, followed by skin (3.3-4.5%), blood (1.4-2.6%), and intestines (1.0-1.7%). One metabolite, glyoxylic acid, which is mutagenic, might be responsible for or contribute to the carcinogenicity of DCA.

Genetic diversity and species diversity of stream fishes covary across a land-use gradient

Genetic diversity and species diversity are expected to covary according to area and isolation, but may not always covary with environmental heterogeneity. In this study, we examined how patterns of genetic and species diversity in stream fishes correspond to local and regional environmental conditions. To do so, we compared population size, genetic diversity and divergence in central stonerollers (Campostoma anomalum) to measures of species diversity and turnover in stream fish assemblages among similarly sized watersheds across an agriculture–forest land-use gradient in the Little Miami River basin (Ohio, USA). Significant correlations were found in many, but not all, pair-wise comparisons. Allelic richness and species richness were strongly correlated, for example, but diversity measures based on allele frequencies and assemblage structure were not. In-stream conditions related to agricultural land use were identified as significant predictors of genetic diversity and species diversity. Comparisons to population size indicate, however, that genetic diversity and species diversity are not necessarily independent and that variation also corresponds to watershed location and glaciation history in the drainage basin. Our findings demonstrate that genetic diversity and species diversity can covary in stream fish assemblages, and illustrate the potential importance of scaling observations to capture responses to hierarchical environmental variation. More comparisons according to life history variation could further improve understanding of conditions that give rise to parallel variation in genetic diversity and species diversity, which in turn could improve diagnosis of anthropogenic influences on aquatic ecosystems.

The Induction of Hepatocellular Neoplasia by Trichloroacetic Acid Administered in the Drinking Water of the Male B6C3F1 Mouse

The prevalence (percent of animals with a tumor) and multiplicity (number of tumors per animal) of hepatocellular neoplasia in the male B6C3F1 mouse exposed to trichloroacetic acid (TCA) in the drinking water were determined. Male mice were exposed to 0.05, 0.5, and 5 g/L TCA for 60 wk (Study 1), to 4.5 g/L TCA for 104 wk (Study 2) and to 0.05 and 0.5 g/L TCA for 104 wk (Study 3). Time-weighted mean daily doses measured for the low, medium, and high dose groups were consistent over the three studies, 6–8, 58–68, and 572–602 mg/kg-d for the 0.05, 0.5, and the 4.5–5 g/L treatment groups, respectively. No significant changes in animal survival were noted across the studies. A significant increase in the prevalence and multiplicity of hepatocellular tumors was found in the 58–68 and 572–602 mg/kg/d TCA dose groups. Nonhepatoproliferative changes (cytoplasmic alterations, inflammation, and necrosis) in mice treated with TCA were mild and dose related. A TCA-induced increase in liver palmitoyl CoA oxidase activity, a marker of peroxisome proliferation, correlated with tumor induction. A linear association was found between peroxisome proliferation and tumor induction. Sporadic increases in the labeling index of nuclei outside of proliferative lesions were observed at carcinogenic doses throughout the studies. Given that there are no compelling data demonstrating genotoxic activity of either TCA or any metabolite, data are consistent with an epigenetic mode of action. The studies provide dose-response data on the development of hepatocellular neoplasia in male mice over a lifetime exposure to TCA. A no-observed-effect-level (NOEL) of 6 mg/kg/d was calculated for neoplastic and nonproliferative liver pathology.

2-Chloroacetaldehyde and 2-chloroacetal are potent inhibitors of DNA synthesis in animal cells.

The effect of 2-chloroacetaldehyde, CAA, a metabolite of vinyl chloride and 2-chloroacetal, CAC, an ethyl diester of chloroacetaldehyde, on DNA synthesis in animal cells has been investigated. Both compounds drastically inhibited DNA synthesis at 10 to 20 microM. The inhibitory effect of the chemicals appears to be directly on DNA synthesis rather than on the uptake of thymidine or the formation of nucleotides. Residual DNA made in the presence of CAA had an average chain length of 300 nucleotides compared to a length of several thousand nucleotides in the absence of CAA. Synchronization experiments revealed that the inhibitory effect is reversible if 2-chloroacetaldehyde is removed within two hours but not after longer exposures.

Fourteen‐day Toxicity Study of 1,3,5‐Trinitrobenzene in Fischer 344 Rats

Toxic effects of 1,3,5‐trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg−1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high‐dose groups (800 and 1200 mg TNB kg−1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg−1 diet) was 92 mg kg−1 BW day−1 for male rats and 80 mg kg−1 BW day−1 for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high‐dose groups was noted. In addition, histopathological examinationsrevealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg−1 BW day−1 was established based on the findings of this study.

Gas Chromatographic and Mass Spectrometric Determination of Hemoglobin Adducts of 1,3-Dinitrobenzene and 1,3,5-Trinitrobenzene in Shrew Cryptotis Parva

1,3-Dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by gas chromatography/mass spectrometry (GC/MS) after either basic (0.1 N NaOH) or acid (2 N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 & pm; 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 & pm; 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 & pm; 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 & pm; 15.3 pg-mg Hb). These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.

Toxicity of Tetryl (N-Methyl-N,2,4,6–Tetranitroaniline) in F344 Rats

The toxicity of tetryl (N-methyl-N,2,4,6–tetranitroaniline) in male and female F344 rats was evaluated after adminstration in the diet for 14 or 90 days. The 14–day study diet concentrations used were 0, 500, 1250, 2000, 2500, and 5000 ppm; the 90–day study diet concentrations were 0, 200, 1000, and 3000 ppm tetryl in the diet. The calculated average daily tetryl intake was 32.1, 82.5, 130.3, 178.9, and 374.4 mg/kg body weight (BW) for females and 31.8, 80.0, 121.0, 170.5, and 349.7 mg/kg BW for males in the 14–day study. For the 90–day studies, the daily tetryl intake was 14.2, 68.8, and 199.0 mg/kg BW for females and 13.0, 62.4, and 179.6 mg/kg BW for males. In the 14–day study, there was a significant decrease in body weights (males), whereas relative(organ/body weight) liver and spleen (females), and kidney (males) weights were significantly increased in the 5000–ppm dose group. Hematological effects observed were decreased hemoglobin and hematocrit and an increased number of reticulocyts in females (2000 to 5000 ppm). Methemoglobin levels in males (2000 to 5000 ppm) and females (5000 ppm) and total blood protein and albumin levels in all groups of males and females (except 500 ppm) were significantly increased. Histopathological changes were observed in kidneys (deposition of cytoplasmic droplets) of all dose groups of male rats. In the sub–chronic (90–day) study, feed intake was reduced in all dose groups, but a significant decrease in terminal body weights was observed in females (1000 and 3000 ppm) and males (3000 ppm). An increase in the relative liver, kidney (1000–3000 ppm), and spleen (3000 ppm) weights were noted in both sexes. The hemoglobin content and red blood cell count were decreased whereas the reticulocyte count was elevated (3000 ppm) in both sexes at 45 and 90 days. Methemoglobin levels were increased in both sexes (1000 and 3000 ppm). Histopathologicalchanges were noted in the spleen (pigment deposition and erythroid cell hyperplasia) of both sexes (3000 ppm) and kidneys (tubular degeneration and cytoplasmic droplets containing alpha-2-micro globulin) of male rats (1000 to 3000 ppm). A no observed adverse effect level (NOAEL) for both sexes was 13 mg/kg BW/day was determined.

Ninety-day toxicity study of sodium monochloracetate in Sprague-Dawley rats

Male and female Sprague-Dawley rats were administered the sodium salt of monochloroacetic acid (SMCA) by oral gavage for a period of 90 consecutive days. Dosage levels of 15, 30, 60 or 120 mg/kg per day were employed. SMCA clearly induced toxicity in both females and males, with the greatest severity in the male animals. Both the liver and kidneys were identified as target organs. At 120 mg/kg per day, 30% of females and 80% of the males died, most within the first 2 days of treatment. Hemorrhagic and congested lungs (possibly a postmortem change) were seen in the early deaths (1-3 days) whereas liver lesions were observed in later deaths. In addition, there was nephrotoxicity as evidenced by elevated creatinine, blood calcium (BCAL), and blood urea nitrogen (BUN) levels. Hepatotoxicity was indicated by increases in the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Both organs showed increased organ-to-body weight ratios. Microscopic examination revealed a significant (P less than or equal to 0.001) increase in chronic renal nephropathy and increased splenic pigmentation at 60 mg/kg per day in the males. Based on the observation of toxicity at all treatment levels in males, a lowest observed adverse effect level (LOAEL) of 15 mg/kg per day is proposed for a 90-day exposure to SMCA by oral gavage to the Sprague--Dawley rat.