Genotoxicity of Lead in Newborn Rat Keratinocytes Exposed In Vitro

Abstract

Cells became resistant to 6-thioguanine (6-TG) when primary monolayer cultures of keratinocytes derived from the skin of newborn rats were exposed to lead acetate (Pb²⁺). The response was measured autoradiographically by the incorporation of [³H]thymidine (TdR) in the presence of 6-TG. In normal cells, 6-TG is converted to a nucleotide by hypoxanthine phosphoribosyl transferase (HPRT). The nucleotide inhibits the replication of DNA and, thereby, prevents mitosis. Resistance to 6-TG can occur in cells in which HPRT activity is absent as a result of mutation or is otherwise inhibited. A dose of 10⁻-⁶ M 6-TG was used to select resistant cells 3 days after their exposure to Pb². At this concentration of the antimetabolite, cells in control cultures (i.e., not exposed to Pb²⁺) dso incorporated [³H]TdR. The number of labeled cells in one-tenth of then “growing area” of each culture was counted and expressed as the number of labeled cells in 100 microscopic fields. The ratio, T/C, between the number of labeled cells in the lead-treated group and in the control group was calculated. When the cells were exposed to Pb²⁺ for 4 hr, the T/C increased with increasing concentration of Pb²⁺ and reached a maximum at a dosage of 3.3 × 10⁻-⁶ M Pb²⁺ in the medium. Because of cytotoxicity, higher dosages resulted in lower T/C. Experiments involving incorporation of [³H]TdR into DNA in the presence of bromodeoxyuridine (BUdR), fluorodeoxyuridine (FUdR), and 6-TG and analysis of DNA by sedimentation in a cesium chloride (CsCl) density gradient showed that in control cultures essentially all the radioactivity was associated with the parental DNA, indicating that repair had occurred, whereas in cultures exposed to Pb²⁺ most of the ³H was in the “heavy” position of the profile, indicating the presence of newly replicated DNA.

These results suggest that resistance to 6-TG in the keratinocyte cultures was a function of Pb²⁺-induced mutation in the HPRT gene. This hypothesis is supported by the finding that the ratio of labeled mitotic figures to total labeled cells was similar in Pb²⁺-treated cultures selected with 6-TG and in untreated cultures not selected with 6-TG. The possibility that resistance is a function of the inhibition of HPRT activity in Pb²⁺-exposed cells is unlikely since Pb²⁺ at the concentration used in these experiments did not inhibit HPRT. Proof that a mutation at the HPRT locus is responsible for resistance to 6-TG in Pb²⁺-exposed keratinocytes still awaits the genetic, immunologic, andor enzymologic demonstration that active HPRT is absent in the resistant cells. This culture technique should be useful in evaluating the potential toxicity of drugs designed for cutaneous use and other environmental chemicals. Since the same protocol can be used with cells from human skin, this technique could also be used to compare toxicologic data from animals to that in human subjects.