Transient inhibition by orotic acid does not abolish the in vivo response of rat hepatocytes to a direct mitogen, lead nitrate

Background: Orotic acid (OA) is able to inhibit hepatocyte proliferation in vivo induced by partial hepatectomy. The present studies were aimed at establishing: (i) whether OA also inhibits hepatocyte proliferation induced by a direct mitogen and, if so (ii) whether the stimulus provided by the mitogen is still expressed following transient inhibition by OA. Methods/Results: In the first experiment male Wistar rats were injected with either lead nitrate (100 μmol/kg, i.v.) or saline and 20 h later some animals receiving the mitogen were also implanted with a 400-mg OA tablet (as OA-methyl ester. i.p.). Multiple injections of 3H-thymidine were given to each rat (50 μCi each, 6 h apart, i.p.) until 2 h before killing. All groups were killed 3 days after the initial treatment. Results indicated that OA almost completely inhibited hepatocyte DNA synthesis and labelling induced by lead nitrate (e.g. labelling index was 1.9±0.5% in the saline-treated group, 44.7±4.0% in the lead nitrate group and 1.4±0.3% in the group receiving lead nitrate + OA). Based on the above results, in a second experiment rats were given a similar dose of lead nitrate and a subset of animals was implanted 20 h later with a 400-mg OA tablet, as previously described. Multiple doses of 3H-thymidine were again given to each rat (20 μCi each, 6 h apart) until 2 h before killing. Animals from both groups were killed at 3, 6 or 8 days after lead nitrate. Results indicated that, while at day 3 lead nitrate-induced DNA synthesis was effectively inhibited by OA, at day 6 the proliferative response was resumed in the group receiving OA. Cumulative labelling index over 6 day was 30.3±1.4 in rats given the mitogen alone and 52.1±2.2 in the group exposed to lead nitrate + OA. Conclusions: These data indicate that: (i) OA is also able to inhibit hepatocyte proliferation induced by a direct mitogen such as lead nitrate; this, in turn, suggests that its inhibitory effect is not unique to the stimulus elicited by partial hepatectomy. (ii) The proliferative response triggered by the mitogen is not abolished by the transient (3–4 days) inhibitory phase imposed by OA. Possible mechanisms underlying these effects are considered in the discussion.