Edith L.C. Lin

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.

Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication

Chronic exposure to trichloroethylene (TCE) results in hepatocellular cancer in mice but not rats. The induction of hepatic tumors by TCE appears to be mediated through nongenotoxic or tumor promotion mechanisms. One cellular effect exhibited by a number of nongenotoxic carcinogens and tumor promoters is the inhibition of gap junction mediated intercellular communication. In the present study, the effects of trichloroethylene (TCE) and its metabolites, trichloracetic acid (TCA), trichloroethanol (TCEth), and chloral hydrate (CH) on gap junction mediated intercellular communication in cultured B6C3F1 mouse and F344 rat hepatocytes were assessed. TCE and TCA inhibited intercellular communication in mouse hepatocytes but not in rat hepatocytes. TCEth and CH had no effect on hepatocyte intercellular communication in either rat or mouse cells. TCE and TCA inhibited intercellular communication in both 24-hr-old and freshly plated mouse hepatocytes. Both compounds produced greater inhibition of intercellular communication in freshly plated cells when compared to 24-hr-old cultures. TCE appeared to require cytochrome P450 metabolism by the mouse hepatocytes to exhibit its inhibitory effect on dye coupling since treatment with SKF-525A prevented the inhibition of intercellular communication by TCE. The inhibitory effect of TCA on intercellular communication was unaffected by treatment with SKF-525A. While the species dependent effect of TCE on intercellular communication may be correlated with different rates and extent of metabolism of TCE by rat and mouse hepatocytes, the inhibiting effect of TCA only on mouse hepatocytes suggests that other intrinsic factors in the male mouse make this species more susceptible to the effects of TCE and TCA on gap junction mediated intercellular communication. These findings may account, in part, for the observed species difference in susceptibility to TCE induced liver carcinogenesis.

Fish Biliary PAH Metabolites Estimated by Fixed-wavelength Fluorescence as an Indicator of Environmental Exposure and Effects

Abstract Biliary polynuclear aromatic hydrocarbon (PAH) metabolites have been studied since the mid 1980s as an indicator of exposure of fish to PAHs. However, the measurements of PAH metabolites are often costly and time-consuming. A simple and rapid method, fixed-wavelength fluorescence (FF), was used to measure the concentrations of benzo(a)pyrene (B[a]P)-type and naphthalene (NAPH)-type PAH metabolites in the bile of brown bullheads ( Ameiurus nebulosus ) collected from Old Woman Creek, Ottawa River, Cuyahoga River-harbor and Cuyahoga River-upstream. The biliary PAH metabolites in fish from the less contaminated Old Woman Creek were significantly lower than those from the industrially contaminated Ottawa and Cuyahoga rivers. The levels of biliary PAH metabolites were found to be related to the PAH sediment contamination for the four sites except Cuyahoga River-upstream, and to the prevalence of fish barbel abnormalities and external raised lesions observed in all rivers except Ottawa. Statistical analysis revealed a significant association between the occurrence of barbel abnormalities and concentrations of biliary NAPH-type metabolites and between the occurrence of raised lesions and concentrations of B[a]P-type metabolites. This study provides added evidence that FF is an effective bile analysis method for determining the exposure of fish to PAHs. This study also indicates that the measurement of PAH metabolites could help establish causal relationship between the chemical exposure and effects such as barbel abnormalities and raised lesions.

Comparison of the effects of acute and subacute treatment of phenobarbital in different strains of mice

A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital (PB), in which diethylnitrosamine (DENA)-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL x C3H) and not affected in C57BL mice. A correlation has been established between the ability of barbiturates and hydantoins to promote tumor formation and their ability to induce liver growth, hepatic DNA synthesis and mixed function oxidase activities. Therefore, we examined in these 3 strains of mice and in C3B6F1 (C3H x C57BL) mice the effect of PB administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by PB in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in the 4-day and 28-day treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with PB, however, after 28 days of treatment there was either a much reduced increase (C57BL and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all 4 types of mice after 4 and 28 days of treatment, PB increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase (AmDm) and 7-ethoxyresorufin-O-deethylase (ErDe) and the oxidation of testosterone (T). The oxidative metabolites of T were similar in the 4 types of mice.

Historical Monitoring of Biomarkers of PAH Exposure of Brown Bullhead in the Remediated Black River and the Cuyahoga River, Ohio

Abstract Biomarkers of exposure to chemical contamination, benzo[a]pyrene (BAP)- and naphthalene (NAPH)-type metabolites, were measured in brown bullhead from a heavily polycyclic aromatic hydrocarbons (PAHs) contaminated section of the Black River, Ohio, during and immediately after remedial sediment dredging in 1990–1991, and in follow-up revisits in 1993 and 1998. Biomarker levels of bullhead from the historically polluted Cuyahoga River and the protected Old Woman Creek National Estuarine Sanctuary in Ohio were also measured over the same time periods. PAH-bile metabolite concentrations of bullheads from the Black River were still elevated in the year following dredging, but were significantly lower in the later resampling years. Metabolite concentrations of Cuyahoga River bullhead decreased significantly between 1991 and 1993, possibly in response to the shutdown of coking operations on the river. Fish from Old Woman Creek showed some variation in metabolite concentrations among periods, but were consistently lower than the other two rivers at each time. Measurement of bile metabolites proved an effective tool for estimating changes in exposure over time and among sampling sites. Trends in biomarkers indicated lowered exposures in the Black and Cuyahoga Rivers. These findings are likely a result of remediation activities in the Black River and source reduction and incidental sediment displacement in the Cuyahoga River.

Glutathione plus cytosol- and microsome-mediated binding of 1,2-dichloroethane to polynucleotides

1,2-[1,2-14C]Dichloroethane was metabolized by rat hepatic microsomes to products that irreversibly bound polynucleotides. The polynucleotides were then enzymatically hydrolyzed and the products separated by a high-performance liquid chromatograph (HPLC) equipped with an ODS or a SCX column. The products of microsome-mediated binding were identified in the HPLC eluate as 1,N6-ethenoadenosine to polyadenylic acid, 3,N4-ethenocytidine to polycytidylic acid, and two cyclic derivatives to polyguanylic acid. 1,2-[1,2-14C]Dichloroethane was also metabolized in the presence of a glutathione (GSH)-cytosolic fraction and a polynucleotide. After enzymatic hydrolysis of the polynucleotide, the major peak of radioactivity was eluted from a Sephadex G-25 column in the salt volume which would exclude the presence of a product containing both GSH and a nucleoside. Chromatography by ODS-HPLC of the major peak from Sephadex G-25 indicated the presence of a GSH metabolite of 1,2-dichloroethane that did not contain a nucleoside. A similar hydrophilic peak was obtained for the hydrolysis products of polynucleotides from a glutathione plus cytosol incubation in which the polynucleotide instead of being added prior to the incubation was added after the incubation. The products of the glutathione plus cytosol metabolism of 1,2-[1,2-14]dichloroethane appear to be glutathione metabolites that coisolated with the polynucleotides rather than covalently bound adducts. In conclusion, covalently bound adducts were identified for microsome-mediated binding of 1,2-dichlorethane to polynucleotides, while no evidence was obtained for glutathione plus cytosol-mediated covalent binding to polynucleotides.

Macromolecular adduction by trichloroacetonitrile in the Fischer 344 rat following oral gavage

Male Fisher 344 rats were administered 1- or 2-[14C]trichloroacetonitrile (TCAN) by oral gavage. DNA was isolated from the liver, kidneys and stomach and several protein fractions (globin, albumin and globulins) were isolated from blood. TCAN binds to both the DNA and the blood proteins in a dose-related manner. More radiolabel was associated with the DNA when the carbon at C2 position was labeled, than at C1 position. However, the position of the radiolabel did not influence the levels of radioactivity associated with the blood proteins. The stomach exhibited the highest level of DNA binding, followed in order by the liver and kidney. TCAN binding level was higher in DNA isolated from rats killed at 24 h than at 4 h after administration. In contrast, the three blood proteins showed similar binding levels, regardless of the exposure time. Radioactivity associated with DNA was not incorporated into the nitrogen bases (i.e. via de novo synthesis) and a covalent binding index (mumol chemical bound/mol nucleotide phosphate per mmol/kg body wt. of chemical administered) of 30-120 was observed for various tissues. Most of the radioactivity (60-80%) associated with globin could be released and separated from the protein by the treatment with concentrated ammonium hydroxide and precipitation of protein by organic solvent. Three peaks were observed in the HPLC elution profiles of the radioactivity released from the globin. Trichloroacetic acid co-eluted with one of these released products (peak II), however, the chemical identity of the material under the major peak (peak III) and peak I are still uncharacterized.