The binding of benzo[a]pyrene to rat liver protein and nucleic acids in vivo

In the present study rats were pretreated with agents that are known to affect the activities of the benzo[a]pyrene (BP) metabolizing enzymes in vitro. Also, agents which are known to alter the levels of hepatic glutathione were used. These experiments were carried out in order to determine the effects of enzyme induction, enzyme inhibition, and glutathione levels on the degree of covalent binding of BP to liver macromolecules in vivo. In addition the roles of aryl hydrocarbon hydroxylase (AHH) and epoxide hydratase (EH) can be studied in this way. Since the degree of covalent binding of polycyclic aromatic hydrocarbons (PAH's) and their tumor-initiating ability have been shown to correlate (13), the factors which govern the extent of covalent binding are of major importance. When ³H-BP was administered intraperitonea11y to male Wistar rats, a certain amount of the compound was bound irreversibly to liver macromolecules. The degree of irreversible binding was found to be dependent on both the dose of BP administered and the time after its injection. The degree of binding was found to be linearly dependent on the dose of BP between the range of 0.125 and 12.5 μ moles, thereby suggesting that the metabolizing pathways of BP were not saturated at these levels. The study showed the maximum level of binding to occur at 12 to 18 hours after 1.25 μ mole of BP, and this fell to 60% of maximum by 48 hours. The BP dosage of 1.25 μ mole was employed throughout the study. The results showed that pretreating rats with SKF 525-A significantly decreased the level of irreversibly bound BP from control levels by about 30%. The decrease in binding after SKF 525-A treatment is in agreement with the evidence that the cytochrome P-450 enzymes are responsible for the activation of BP to reactive intermediates, and that they can be inhibited by this compound (42). SKF 525-A at 35 mg/kg, a dose which produces inhibition of AHH in vitro (74) did not decrease the binding of BP in vivo. SKF 525-A at 50 mg/kg or higher was required to produce a decrease in binding, indicating the necessity to reach a higher effective hepatic concentration of SKF 525-A to inhibit the irreversible binding of BP in vivo, in contrast to a lesser amount of SKF 525-A required to inhibit BP hydroxylase in vitro. Oral methadone pretreatment failed to alter the level of BP binding to liver macromolecules. Methadone was found to increase hepatic epoxide hydratase by 212% in male Wistar rats (66), but in the present study this did not have any influence on the degree of binding of BP in vivo. 3-Methy1cholanthrene (3~MC) pretreatment was found to significantly decrease the level of irreversibly bound BP from control by about 30%. The possible causes of this 3-MC induced decrease in binding are discussed. One possibility is that the 3-MC induced decrease could be due to an alteration in the pathways of BP metabolism. The existence in the liver of various forms of cytochrome P-450 (39) along with the evidence that 3-MC induces a spectrally distinct cytochrome P-448 (40,58) could suggest that 3-MC alters BP metabolism to sites on the BP molecule that produce less reactive intermediates, and thereby decreases the degree of binding. Neither xysteine nor diethyl maleate pretreatment altered the level of irreversibly bound BP from control. The data obtained from these experiments can be explained by one or more of the following mechanisms: no significant depletion of glutathione by BP occurs, there is a lack of a glutathione threshold level for binding to take place, or there is a drastically different role for glutathione than its role in the protection of hepatic macromolecules from alkylation by active metabolites of acetaminophen. The enzyme-mediated binding of BP to liver macromolecules in vitro and its inhibition by methadone, SKF 525-A, 3-MC, glutathione, and cysteine was demonstrated and the relevance of these findings towards the present experiments was discussed. Throughout the study a second population of animals showed binding of BP that was both qualitatively and quantitatively different from the first. The percentage of animals that fell into this 2nd population was 19% (46 out of 250) of all the animals used in the study.Pharmaceutical Sciences, Faculty ofGraduat