Induction of hepatic microsomal drug-metabolizing enzymes by inhibitors of 5-lipoxygenase (5-LO): studies in rats and 5-LO knockout mice.

The effect of 5-lipoxygenase (5-LO) inhibitors on the hepatic microsomal mixed-function oxidase (MFO) system of rodents was investigated. After establishing the relative in vitro and in vivo potencies of the 3 test compounds, male Crl:CD (SD) BR rats received CJ-11,802 (0, 10, 50, or 200 mg/kg/day), zileuton (0, 10, 60, or 300 mg/kg/day) or ZD2138 (0 or 200 mg/kg/day) once daily by oral gavage for 14 (zileuton and ZD2138) or 30 (CJ-11,802) consecutive days. Controls were given an equivalent volume of 0.5% methylcellulose vehicle. At necropsy, all livers were weighed, and sections from representative animals (control and highest dose for each compound) were utilized to prepare hepatic microsomal fractions, which were assayed for cytochrome P-450 (CYP) content and the activities of cytochrome c reductase (CRed), para-nitroanisole O-demethylase (p-NOD), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-dealkylase (PROD). A dose-related increase in liver weight occurred in rats given CJ-11,802 and zileuton, while animals administered ZD2138 were unaffected. Rats given CJ-11,802 (200 mg/kg/day) and zileuton (300 mg/kg/day) had increases in CYP, EROD, PROD, CRed and p-NOD compared to corresponding controls, while only the latter two activities were elevated in animals administered ZD2138. To determine if induction of the hepatic microsomal MFO system was related to 5-LO inhibition, male DBA wild-type and 5-LO knockout mice were administered either CJ-11,802 (200 mg/kg/day) or vehicle by oral gavage for 14 consecutive days. At necropsy, liver weight, CYP content, and CRed activity were measured and all were increased similarly in the treated wild-type and knockout mice compared to corresponding controls, indicating that induction was not related to inhibiting 5-LO.

The use of explant lens culture to assess cataractogenic potential.

Explanted cultures of crystalline lenses have been used to investigate mechanisms of xenobiotic-induced cataract formation. However, very few studies have utilized mechanistic information to predict the cataractogenic potential of structurally diverse xenobiotics. The present investigation outlines how visual assessment of lens clarity, biochemical endpoints of toxicity, and mechanisms of lenticular opacity formation can be used to select compounds with a lower probability of causing cataract formation in vivo. The rat lens explant culture system has been used to screen thiazolidinediones against ciglitazone for their direct cataractogenic potential in vitro. The two compounds that were selected as development candidates (englitazone and darglitazone) did not produce cataracts in rats exposed daily for 3 months. The culture system has also been used to illustrate that the lens is capable of metabolizing compounds to reactive intermediates. In this example, the toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a model cataractogen, was attenuated by inhibiting lenticular cysteine conjugate beta-lyase metabolism using aminooxyacetic acid. Finally, this model was used retrospectively to investigate the cataractogenic potential of CJ-12,918 and CJ-13,454 in rats. These compounds showed differences in the incidence of cataract formation in vivo based on differences in hepatic metabolism and penetration of parent drug and metabolites into the lens. The rank order of cataractogenic potential in vitro correlated better with in vivo results when an induced S9 microsomal fraction was added to the culture media. However, the model did not correctly predict the cataractogenic potential of ZD2138, a structurally similar compound. These studies illustrate the use of explant culture to assess mechanisms of cataract formation and outline its use and limitations for predicting cataractogenic potential in vivo.

Acetaminophen nephrotoxicity in CD-1 mice. I. Evidence of a role for in situ activation in selective covalent binding and toxicity.

Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr-fasted, 3-month-old male CD-1 mice results in necrosis of the renal convoluted proximal tubules. To selectively inhibit APAP activation in the kidney but not in the liver, 10-week-old male CD-1 mice were castrated under ether anesthesia and allowed to recover for a minimum of 2 weeks before use. Microsomal activation of APAP in vitro was monitored by trapping the APAP-derived electrophile as an N-acetylcysteine conjugate. Production of the conjugate was significantly decreased in renal microsomes from castrated mice. By contrast, hepatic microsomal production of the APAP conjugate was not changed by castration. Castration also did not alter APAP covalent binding in liver or APAP hepatotoxicity. Also, the overall metabolism of APAP was not altered by castration, as there were no significant differences in the 24-hr excretion of APAP urinary metabolites between castrated and sham-operated mice. However, castration did significantly protect mice against APAP nephrotoxicity, as evidenced by decreases in plasma urea nitrogen levels and in the severity of histopathologic damage assessed 12 hr after APAP. Castration also reduced the level of selective covalent binding of APAP metabolites to renal proteins as detected on Western blots with affinity-purified anti-APAP antibodies. These findings suggest that, for the mouse, intrarenal biotransformation of APAP to a reactive electrophile significantly contributes to the APAP covalent binding within the kidneys and the subsequent nephrotoxicity.

Evidence against deacetylation and for cytochrome P450-mediated activation in acetaminophen-induced nephrotoxicity in the CD-1 mouse.

Acetaminophen (APAP) administration (600 mg/kg, po) results in proximal tubular necrosis in 18-hr fasted, 3-month-old male CD-1 mice. This study was undertaken to determine if deacetylation of APAP to p-aminophenol (PAP) is a prerequisite to nephrotoxicity in the mouse, as it is in the Fischer rat. Administration of either APAP or PAP to mice resulted in significant elevations of plasma urea nitrogen and marked proximal tubular necrosis at 12 hr after dosing. Prior inhibition of APAP deacetylation by the carboxylesterase inhibitors bis(p-nitrophenyl) phosphate or tri-o-tolyl-phosphate did not alter APAP hepatotoxicity or nephrotoxicity. By contrast, pretreatment with the MFO inhibitor piperonyl butoxide decreased APAP nephrotoxicity but not that of PAP. Immunochemical analysis of kidneys from APAP-treated mice demonstrated covalently bound APAP but no binding was detected after mice were treated with a nephrotoxic dose of PAP. Since the antibody used has been characterized as being directed primarily against the N-acetyl moiety of bound APAP metabolite and since it did not react with kidney proteins of mice given a nephrotoxic dose of PAP, it is unlikely that APAP deacetylation preceded binding or that acetylation of bound PAP occurred. Taken together, these findings indicate that in the CD-1 mouse, APAP-induced nephrotoxicity differs from that previously described for the Fischer rat and likely involves cytochrome P450-dependent activation and subsequent covalent binding of a metabolite without prior deacetylation.

Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: an in vivo and in vitro analysis.

Acetaminophen (APAP) administration (600 mg/kg, po) to fasted male CD-1 mice resulted in cellular damage to liver, lung, and kidney. An affinity purified antibody against covalently bound APAP was used to identify APAP-protein adducts in microsomal and cytosolic extracts from these target organs. The proteins were resolved on SDS-PAGE, transblotted to nitrocellulose membranes, and analyzed immunochemically. Covalent binding of APAP to intracellular proteins was only observed in those organs which exhibited cellular damage; no APAP adducts were detected in tissues which did not undergo necrosis. In all target tissues the arylation of proteins was not random but highly selective with two adducts of 44 and 58 kDa accounting for the majority of the total APAP-bound proteins which were detected immunochemically. In addition, a third major APAP-protein adduct of 33 kDa was also observed in kidney cytosol. The severity of tissue damage and the amount of adducts present in these tissues could be significantly reduced when mice were pretreated with the mixed function oxidase inhibitor, piperonyl butoxide, prior to APAP dosing. Immunochemical analysis of plasma from APAP-treated animals indicated the presence of several protein adducts by 4 hr following drug administration. These adducts did not appear to be of plasma origin. Incubation of cytosolic proteins from liver, lung, kidney, spleen, brain, and heart with an APAP metabolite generating liver microsomal system demonstrated that the cytosolic 58-kDa protein target was native to all tissues tested. By contrast, the 58-kDa protein target did not appear to be endogenous to plasma since it was not detected when plasma was incubated in vitro with the liver microsomal system. These studies indicate that, although the 58-kDa proteins appear to be endogenous to both target and nontarget tissues, the 58-kDa APAP-protein adducts are detectable only in tissues which become damaged by APAP.

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice.

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.

Acetaminophen-induced inhibition of hepatic mitochondrial respiration in mice.

Morphological changes are observed in mitochondria early in the course of acetaminophen (APAP) hepatotoxicity. In order to determine if functional deficits also occur, this study examined the effect of APAP, in vivo and in vitro, on mitochondrial respiration in fasted, male CD-1 mice (3-4 months old). After a hepatotoxic dose of APAP (600 mg/kg, po), when glutamate was used as the respiratory substrate, state 3 respiration (ADP-stimulated) was inhibited and this was reflected in a decreased respiratory control ratio (RCR). In contrast, when succinate was the respiratory substrate, the decreased RCR was reflective of an increase in state 4 (resting) respiration. There was no detectable effect after a nonhepatotoxic dose of APAP (300 mg/kg, po). These APAP-induced respiratory effects and hepatotoxicity were prevented by piperonyl butoxide pretreatment, and were absent in 1- and 2-month-old mice, which are resistant to APAP-induced damage. Since the APAP-induced inhibition of mitochondrial respiration, in vivo, correlated with age-related and piperonyl butoxide-dependent differences in toxicity, the data suggest that the in vivo effects result, at least in part, from a mixed-function oxidase generated metabolite. In vitro, both state 3 and state 4 respiration, as well as the RCR, were inhibited by APAP in a concentration-dependent manner with glutamate as substrate. However, no effects were observed with succinate as substrate, thereby contrasting with results obtained following in vivo exposure. Therefore the in vitro effects of APAP are different from those observed in vivo and may result from a direct insult of the parent compound. These studies suggest that early alterations in mitochondrial function may be mechanistically important in APAP hepatotoxicity.

l-alpha-Acetylmethadol administration to lactating rat dams. Effect on hepatic aniline hydroxylase and ethylmorphine N-demethylase activities in rat pups.

l-alpha-Acetylmethadol (LAAM) was administered to lactating rat dams, and subsequent effects on hepatic drug-metabolizing enzymes of their offspring were assessed. Dams were given LAAM or a control solution in their drinking water following parturition and throughout lactation. Hepatic ethylmorphine-N-demethylase (EMDM) and aniline hydroxylase (AH) activities, as well as cytochrome P-450 content, were determined in the offspring at 21-23 days of age, or following sexual maturation (61-64 days). LAAM induced AH and EMDM activities, as well as cytochrome P-450 content in both male and female 21-23-day-old pups compared to controls; these differences were not observed at 61-64 days of age. In addition, normal sex-related differences in EMDM activity were apparent at 61-64 days of age. These results demonstrate the LAAM administration to lactating dams causes hepatic metabolic induction in the sexually immature rat, suggesting that LAAM and/or its metabolites passed to the pups via the milk. These changes, induced by LAAM administered via lactation, are reversible and do not interfere with the normal development of sex-dependent differences in hepatic EMDM activity observed in rats following sexual maturation.

Changes in phase I and phase II biotransformation with age in male Fischer 344 rat colon: relationship to colon carcinogenesis.

Using male Fischer 344 rats classified as young (2-4 months), middle aged (12-14 months), and old (22-25 months), the activities of several Phase I and Phase II biotransformation pathways in the large intestine were investigated, including benzo[a]pyrene hydroxylase (BPOH), alcohol dehydrogenase (ADH), glutathione S-transferase (GST), glutathione peroxidase (GSH-PX), beta-glucuronidase (BG), and microsomal and nuclear glucuronyltransferase (UDPGT). Levels of oxidized (GSSG) and reduced (GSH) glutathione and uridine 5'-diphosphoglucuronic acid (UDPGA) were also measured. BPOH increased 33% in old rats, while ADH and BG activity remained unchanged with age. Nuclear UDPGT remained unchanged with age, whereas form I of GSH-PX declined slightly in old rats. GST, microsomal UDPGT, and form II of GSH-PX declined by 38, 37 and 44%, respectively, in old rats. The decrease in GST and microsomal UDPGT was also significant in middle aged rats. Levels of colonic GSH, GSSG and UDPGA were found to be unchanged with age. These in vitro data suggest the possibility that if reactive intermediates are generated to the same extent in old rats as in young rats, decreased detoxification mechanisms in the old rat may increase susceptibility of the colon to actions of chemical carcinogens.

Age-related increased susceptibility of male Fischer 344 rats to acetaminophen nephrotoxicity.

Male Fischer 344 rats classified as young (2-4 months), middle-aged (12-14 months) and aged (22-25 months) received 300, 600 or 800 mg/kg acetaminophen (APAP) intraperitoneally and were sacrificed 24 hr later. Blood urea nitrogen (BUN) concentration and urinary glucose and osmolality were determined. In addition, kidneys were evaluated for histopathological changes. APAP did not affect osmolality or BUN concentrations and failed to produce lesions after any dose in young rats. Osmolality was decreased 40% and 50% in middle-aged and aged rats, respectively, after 800 mg/kg APAP. Glucosuria was prominent in aged rats after the 600 and 800 mg/kg doses were administered, while middle-aged rats showed little glucosuria after these doses. BUN concentrations were elevated 89% and 183% in middle-aged and aged rats, respectively, given 600 mg/kg APAP; after 800 mg/kg, BUN concentrations were elevated approximately four-fold in both age groups. Pathological evaluations showed a greater incidence of acute tubular necrosis (ATN) in aged kidneys compared to kidneys of middle-aged rats after 600 mg/kg, while the two older groups exhibited similar, more severe ATN after 800 mg/kg APAP. These data suggest an age-related increased susceptibility of male Fisher 344 rats to APAP nephrotoxicity.

The development of acetaminophen-induced nephrotoxicity in male Fischer 344 rats of different ages.

Male Fischer 344 rats classified as young (2-4 months), middle-aged (12-15 months) and aged (22-25 months) were administered 600 mg/kg acetaminophen (APAP) IP. Rats were killed 6 and 12 h after dosing, and renal damage evaluated by blood urea nitrogen (BUN) levels and histopathology. In addition, plasma levels of APAP and its sulfate and glucuronide conjugates were determined after 6 h. There was no evidence of renal damage in any age group 6 h after APAP. While no nephrotoxicity was present in young animals after 12 h, BUN was elevated 94% and 214% in middle-aged and aged rats, respectively, compared to young animals. At 12 h, APAP-induced renal lesions were more severe in aged rats compared to middle-aged animals. APAP-induced renal damage, as judged by BUN and histopathology, was not altered in young or middle-aged rats following unilateral nephrectomy. Six hours after APAP, both the middle-aged and aged animals had significantly higher plasma levels of APAP and APAP glucuronide compared to young rats. There were similar amounts of the sulfate conjugate in the plasma of each age group. This suggests pharmacokinetic differences could contribute to the age-related increased susceptibility of male Fischer 344 rats to APAP-induced nephrotoxicity.