Journal of Hepatology 1997; 26: 203-208 Printed in Denmark All rights reserved Munksgaard Copenhagen Copyright 0 European Association for the Study of the Liver 1997 Journal of Hepatology ISSN 0168.8278 zyxwvutsrqponmlk Transient inhibition by erotic acid does not abolish the in viva response of rat hepatocytes to a direct mitogen, lead nitrate Ezio LaconirT2, Aroon Yusuf3, Ali R. Jahangir3, Sergio Laconi’, Prema M. Rao3, Srinivasan Rajalakshmi3, Francesca Sanna’, Paolo Pani’, Antonio Monni2 and Dittakavi S. R. Sat-ma3 ‘Istituto di Patologia Sperimentale, University of Cagliari, and 20spedale Oncologico “A. Businco”USL 21, Cagliari, Italy, and 3Department of Pathology, University of Toronto, Toronto, Canada Background: Orotic acid (OA) is able to inhibit hepatocyte proliferation in viva induced by 2/3 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 pro- vided by the mitogen is still expressed following transient inhibition by OA. Methods/Results: In the first experiment male Wis- tar rats were injected with either lead nitrate (100 pmol/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.). Mul- tiple injections of 3H-thymidine were given to each rat (50 @i each, 6 h apart, i.p.) until 2 h before killing. All groups were killed 3 days after the ini- tial treatment. Results indicated that OA almost completely inhibited hepatocyte DNA synthesis and labelling induced by lead nitrate (e.g. labelling index was l.tiOS% 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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC 0 ROTIC acid (OA) is a normal precursor of pyrimi- dine nucleotides and is able to promote the car- cinogenic process in different systems, including rat liver (l-8). The mechanism underlying its promoting activity has been particularly investigated in the rat liver model (9-17). It was recently shown that OA Received 15 February; revised 14 May: accepted 15 May 1996 Correspondence: Dr. Ezio Laconi, Istituto di Patologia Sperimentale, Via Porcell4,09125 Cagliari, Italy. Tel: 070-668642. Fax: 070-662574. OA tablet, as previously described. Multiple doses of 3H-thymidine were again given to each rat (20 j&i each, 6 h apart) until 2 h before killing. Ani- mals 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 prolifera- tive response was resumed in the group receiving OA. Cumulative labelling index over 6 days was 30.3k1.4 in rats given the mitogen alone and 52.1ti.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 hepa- tectomy. (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 consid- ered in the discussion. Key words: Cell proliferation; Hepatocyte; Inhibi- tion; Mitogenic stimulus; Orotic acid. can inhibit normal hepatocyte proliferation induced both in vitro in response to a variety of growth factors (10-13) and in vivo following 2/3 partial surgical hepatectomy (PH) (14). Moreover, rat hepatocyte nodules developing during carcinogenesis were found to be relatively resistant to the mitoinhibitory effect of OA (14-16) thus suggesting that differen- tial mitoinhibition is likely to be a component during promotion by this agent (14-17). In light of these findings, it becomes important to elucidate the bases for the OA-induced mitoinhibition. 203