| Based on reports suggesting that hydrocortisone (HYD) increased N-acetylation in the rabbit, we examined the potential of glucocorticoids (dexamethasone, DEX, prednisolone, PRED and HYD) to regulate NAT activity in the liver of male Sprague-Dawley rats (S-D). Repeated intraperitoneal administration of all three agents to rats for 10 days resulted in a modest increase in the rate of procainamide acetylation (ex-vivo). When normalized to cytosolic protein content, the potency of induction was PRED $>$ DEX $>$ HYD (30, 29 and 18% increase, respectively), while normalization to liver weight demonstrated equipotent NAT induction by the three agents ($sim$40%).;The problem in studying alterations in NAT activity in response to non-genetic factors has been limited by the availability of a viable in vitro model. Therefore, we examined the utility of primary cultures of adult male S-D rat hepatocytes as a potential approach in circumventing this problem. We characterized (1) the time course of NAT activity in rat hepatocytes on different matrices; (2) optimized conditions for measuring activities and (3) through a series of biochemical studies (thermostability, MTX, VFK) confirmed that both enzymes were expressed throughout the culture period. Furthermore, using Northern blot analysis we qualitatively demonstrated the presence of both mRNAs encoding for NAT1 and NAT2 isozymes on days 0, 3 and 6 of the culture. However, both mRNAs were absent or barely detectable on day 1.;Direct determination of NAT activity in individual culture plates before (48 hr after seeding the cells) and after treatment with DEX $(10sp{-9}, 10sp{-8}, 10sp{-7}, 10sp{-6}$ M; days 2-4 of culture) was not a valid approach, since residual arylamine (parent and/or metabolites) from incubations conducted before treatment were still present on day 4 of culture. We reexamined NAT responsiveness to DEX by comparing the mean concentrations of AcPABA and AcSMZ between control and treated plates on day 4 of the culture only. No significant difference in the mean of AcSMZ concentrations (triplicate incubations) between control and treatment groups was found. Surprisingly, AcPABA concentrations from control and treatment groups were below the level of detection and/or quantification. This phenomenon was most likely due to binding of AcPABA to Matrigel$spcircler$ as suggested from our data. Finally, treatment of hepatocytes for 2 days with 10$sp{-7}$ DEX resulted in a 5-fold increase in TAT activity and was significant when compared to control (p $<$ 0.05). Moreover, increasing the time of treatment exposure from 2 to 3 days produced a 7-fold increase in the latter enzyme when compared to control activity. This suggests that primary cultures of hepatocytes were responsive to DEX.;Hence, primary cultures of rat hepatocytes appear to be a viable model for studying NAT regulation. However, the modest induction of NAT observed after glucocorticoid pretreatment in vivo was not observed in cultured hepatocytes. They are, however, responsive to glucocorticoids, as demonstrated by the induction of TAT. |
