Mutagenicity and metabolism of dimethyl phthalate and its binding to epidermal and hepatic macromolecules

Abstract

As an active ingredient in insect repellents, dimethyl phthalate (DMP) had previously been shown to produce chromosomal aberrations in the livers of rats following subchronic application of the phthalate to skin. When we tested DMP in the Ames mutagenesis assay, it produced in bacterial tester strain TA100 (but not TA98) a dose-related mutagenic response that was abolished by NAD- and NADP-independent metabolism associated with rat liver microsomal preparations (S9). In a host-mediated mutagenesis assay, rats were injected ip with DMP (2 g/kg body weight); urine was collected for 24 h, extracted, and analyzed for mutagenic activity and phthalic acid-containing derivatives. The extracted urine was not mutagenic to TA100 and contained an equivalent of 1.96 mg phthalate/ml urine. More than 97% of the phthalic acid-containing derivatives present in the extracted urine consisted of the nonmutagenic metabolite of DMP, monomethyl phthalate (MMP). In vitro experiments showed that rat liver homogenates hydrolyzed 93% of carbonyl-labeled 14C-DMP (7.7 mM) to MMP in 2 h and bound 0.07 nmol of [14C]phthalate/mg liver macromolecules. By contrast, rat epidermal homogenates metabolized only 5% and bound 38-fold higher levels of carbonyl-labeled 14C-DMP (2.66 nmol/mg of macromolecules), with no detectable binding to nucleic acids. Compared to epidermis and plasma, liver had a fivefold higher rate of DMP monoesterase activity (1240 nmol/h/mg protein), which, when inhibited by 67%, resulted in a 4.4-fold increase in phthalate-bound hepatic macromolecules (0.31 vs. 0.07 nmol of carbonyl-labeled 14C-DMP/mg macromolecules). In addition to MMP, formaldehyde was produced during the metabolism of DMP by liver. When ethanol was used to inhibit the oxidation of DMP-derived methanol by hepatic homogenates, there resulted a 74% reduction in the accumulation of formaldehyde and similar reductions of 71 and 73% in the binding of methyl-labeled 14C-DMP to nucleic acids and macromolecules. (Methyl-labeled, unlike carbonyl-labeled, 14C-DMP yields a 14C-labeled methanol when hydrolyzed.) These results indicate that the DMP diester is a weak bacterial mutagen, which binds to epidermal and hepatic macromolecules other than nucleic acids, and that although the rapid hepatic metabolism of DMP to its monoester (MMP) and methanol affords protection against higher levels of phthalate binding as well as against DMP-induced bacterial mutagenesis, it also oxidizes DMP-derived methanol to formaldehyde, a metabolite that binds macromolecules, including nucleic acids.