Metabolite screening of aromatic amine hair dyes using in vitro hepatic models.

Aromatic amines and heterocyclic amines are widely used ingredients in permanent hair dyes. However, little has been published on their potential for oxidation via hepatic cytochrome P450s. Therefore, the authors screened nine such compounds for their potential to undergo oxidative metabolism in human liver microsomes. Toluene-2,5-diamine (TDA), p-aminophenol, m-aminophenol, p-methylaminophenol, N,N'-bis(2-hydroxyethyl)-p-phenylenediamine, and 1-hydroxyethyl-4,5-diaminopyrazole showed no evidence of oxidative metabolism. Oxidized metabolites of 4-amino-2-hydroxytoluene (AHT), 2-methyl-5- hydroxyethylaminophenol (MHEAP), and phenyl methyl pyrazolone (PMP) were detected, but there was no evidence of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent covalent binding to microsomal protein, suggesting that these are not reactive metabolites. Metabolism of AHT, MHEAP, PMP, and TDA was further studied in human hepatocytes. All these compounds underwent conjugation, but no oxidative metabolites were found. The results suggest that none of the hair dye ingredients tested showed evidence of hepatic metabolism to potentially biologically reactive oxidized metabolites.

Antihistamine-containing cough/cold medications present a low hazard in pediatric accidental exposure incidents: analysis of Poison Control Center data.

We studied accidental exposure to pediatric cough/cold medications in children under 6-y-of-age to determine whether the presence of an antihistamine (chlorpheniramine) in the product increased the likelihood for adverse outcomes. General accidental exposure cases reported to the American Association of Poison Control Centers (AAPCC) during 1988-1992 were analyzed for specific over-the-counter cough/cold pediatric products containing identical concentrations of active ingredients except for the presence or absence of chlorpheniramine. These case reports were evaluated for differences in medical outcome, symptom assessment, management site and therapy, as coded by poison control centers participating in the AAPCC Toxic Exposure Surveillance System. A total of 10,289 cases of accidental exposures were evaluated for the specific products included in this analysis. While these cases represented a small percentage of total exposures to these products (approximately 3% of total cases in children under 6-y-of-age reported to the AAPCC for all cough/cold medications during 1988-1992), they provided a unique opportunity to evaluate the impact of the antihistamine component of the formulation. This study demonstrated that the presence of chlorpheniramine did not affect the medical outcome or the extent to which symptoms were reported. Additionally, similar percentages of exposures for these 2 products were managed at the site of the incident, and required either no therapy or only required fluids and observation. There were no notable differences in the percentage of cases which involved more aggressive treatment procedures (activated charcoal, cathartic, lavage). This analysis demonstrates that over-the-counter cough/cold medications containing chlorpheniramine present low potential for hazard in cases of accidental ingestions in young children and do not show an increased likelihood for adverse outcomes of accidental exposures compared to cough/cold medications not containing this antihistamine.

Effects of doxylamine succinate on thyroid hormone balance and enzyme induction in mice.

The effects of doxylamine (as the succinate salt) on microsomal enzyme activity and serum thyroid hormone levels were examined in B6C3F1 mice following dietary exposure for 7 or 15 days (0, 40, 375, 750, or 1500 ppm in diet, expressed as free base doxylamine). In addition, the hepatic P450 enzyme inducer sodium phenobarbital (375 ppm, expressed as free acid phenobarbital) was used as a positive control for CYP2B induction. Exposure of mice to doxylamine produced dose-related increases in liver weight at both time points. Liver weights were also increased in the phenobarbital-treated mice. Doxylamine treatment caused a dose-dependent increase (up to 2.6-fold) in liver microsomal cytochrome P450 in both male and female mice, at both time points. Analyses of the activities of various hepatic microsomal cytochromes P450 indicated that doxylamine caused a marked induction of CYP2B enzymes. This was demonstrated by a large increase in the O-dealkylation of 7-pentoxyresorufin (up to 38-fold) and the 16beta-hydroxylation of testosterone (up to 6.9-fold), both of which are indicative of CYP2B induction. In addition, like phenobarbital, doxylamine treatment resulted in a modest induction of CYP3A and CYP2A enzymes and approximately a 50% increase in thyroxine-glucuronosyltransferase activity. Doxylamine did not appear to induce P450 enzymes in the CYP1A, CYP2E, or CYP4A enzyme subfamilies. None of the enzyme-inducing effects of doxylamine could be distinguished from those of phenobarbital. These results suggest that doxylamine is a phenobarbital-type inducer of liver microsomal cytochrome P450 in B6C3F1 mice. Exposure to either doxylamine or phenobarbital also resulted in decreases in serum thyroxine (T4) levels (approximately 80% of control) with compensatory increases in serum thyroid-stimulating hormone levels (approximately 4-fold). No clear changes in serum triiodothyronine levels were apparent. These findings are consistent with the hypothesis that doxylamine increases the activity of those hepatic enzymes involved in T4 metabolism.

Assessment of doxylamine influence on mixed function oxidase activity upon multiple dose oral administration to normal volunteers.

The primary purpose of this study was to assess the influence of doxylamine and phenobarbital on antipyrine/metabolites pharmacokinetics and 6 beta-hydroxycortisol urinary excretion. This study was conducted in 48 healthy male human volunteers (16 per treatment group) using a parallel study design. Treatment groups consisted of 12.5 mg of doxylamine succinate, placebo, or 30 mg of phenobarbital administered orally every 6 h for 17 days. Results indicate that no statistically significant differences were observed between the doxylamine and placebo groups that are indicative of enzyme induction. For the phenobarbital group, a significant increase for antipyrine total (36 versus 45 mL/h/kg) and nonrenal (35 versus 44 mL/h/kg) clearances and 6 beta-hydroxycortisol excretion (338 versus 529 micrograms) and a significant decrease in the terminal exponential half-life (11 versus 9 h) of antipyrine were observed.

Evaluation of olestra in short-term genotoxicity assays.

Olestra, a mixture of hexa-, hepta- and octa-esters formed from the reaction of sucrose with long-chain fatty acids, was evaluated for its genotoxic potential in the Salmonella/mammalian microsome test, the L5178Y thymidine kinase (TK+/-) mouse lymphoma assay, an unscheduled DNA synthesis assay in primary rat hepatocytes, and an in vitro cytogenetic assay in Chinese hamster ovary cells. The results indicated that olestra was non-genotoxic in these assays.

Genotoxicity of zinc in 4 short-term mutagenicity assays.

The genotoxicity of zinc was examined in 4 short-term mutagenicity assays. Zinc acetate produced dose-related positive responses in the L5178Y mouse lymphoma assay and an in vitro cytogenetic assay with Chinese hamster ovary cells, but was negative in the Salmonella mutation assay and did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. Zinc-2,4-pentanedione produced frameshift mutations in Salmonella tester strains TA1538 and TA98, but did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. The effect of ligand binding of zinc in the in vitro test systems is discussed.

DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography.

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.

Lack of DNA-strand breaks in rat testicular cells after in vivo treatment with sodium fluoride.

Oral administration of up to 84 mg/kg of NaF to adult male rats did not induce DNA-strand breaks in testicular cells when measured by alkaline elution. Although plasma fluoride levels were as high as 12 ppm is rats given 84 mg/kg of NaF, testicular fluoride levels in most cases were only 10-20% of plasma levels. Fluoride did not accumulate in the testes after 5 daily treatments. Therefore, it is unlikely that NaF, even at high doses, poses a hazard with respect to heritable genetic effects.

Lack of specific inhibition of DNA repair in WI-38 human diploid fibroblasts by sodium saccharin.

The ability of sodium saccharin (NaS) to inhibit the repair of DNA damaged by UV irradiation was examined in cultured WI-38 human diploid fibroblasts. Cesium chloride density gradient ultracentrifugation was used to measure DNA repair and DNA replication. NaS (10-10,000 micrograms/ml) did not specifically inhibit UV light-induced DNA repair. At doses of NaS (1785 and 10,000 micrograms/ml) that caused a 62-67% inhibition of semiconservative DNA replication, there was little or no inhibition of DNA repair synthesis. In cell cultures not exposed to UV irradiation, NaS failed to induce DNA repair. RNA synthesis and protein synthesis were unaffected by NaS at all doses tested. The inhibition of semiconservative DNA replication at higher doses of NaS may be a manifestation of cytotoxicity. In contrast to results with NaS, WI-38 cells were very sensitive to DNA repair inhibition by the well-studied inhibitor quinacrine-HCl. These results do not support mechanisms of saccharin-induced tumorigenesis involving either direct induction of DNA damage or inhibition of the repair of DNA damage caused by other agents.

Validation of an in vivo alkaline elution assay to detect DNA damage in rat testicular cells.

An alkaline elution assay was used to measure DNA damage in the rat testis after in vivo treatment with chemicals. All of the chemicals reported to induce heritable mutations in a mammalian assay (mouse specific locus test) were positive in the rat alkaline elution assay. Chemicals that are negative or inconclusive in the specific locus test did not cause detectable DNA damage in rat testes. DNA damage was detected by alkaline elution after either intraperitoneal or oral administration of chemical mutagens. Data from these validation studies were used to establish criteria to be used for evaluation of alkaline elution results with materials of unknown potential for genotoxic effects in germinal tissue.

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens.

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.

Alkaline elution of rat testicular DNA: detection of DNA strand breaks after in vivo treatment with chemical mutagens.

The alkaline elution technique was used to measure DNA strand breaks in rat testes after intraperitoneal injection of several chemicals known to cause heritable mutations in rodents. Methyl methanesulfonate (MMS), ethyl methanesulfonate, methylnitrosourea, and ethylnitrosourea all produced single strand breaks in rat testicular DNA. For both of these pairs of homologous alkylating agents the relative potency was methyl analog greater than ethyl analog. Strand breaks produced by MMS appeared rapidly (within 2 h) in rat testicular DNA and were partially repaired within 24 h. Studies with low doses of MMS indicate that the assay has the sensitivity to detect DNA strand breaks in the testis after a dose of only 5 mg/kg. Variability in DNA elution profiles for individual control animals and for individual animals given identical doses of MMS was small. In contrast to the results with known mutagens, intraperitoneal injection of nonmutagens such as dimethyl sulfoxide, phenol, and Triton X-15, did not produce strand breaks in testicular DNA. These data indicate that this assay detects DNA strand breaks in the rat testis. The results for several heritable mutagens and nonmutagens are qualitatively predictive of mutagenic activity in the testis.