Terry V. Zenser

Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites: pH stability and synthesis by human and dog liver

Glucuronide conjugates of arylamines are thought to be important in the carcinogenic process. This study investigated the pH stability and synthesis of glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites by human and dog liver. Both dog and human liver slices incubated with 0.06 mM [3H]-4-aminobiphenyl produced the N-glucuronide of 4-aminobiphenyl as the major product. After 2 hr of incubation, the N-glucuronide of 4-aminobiphenyl represented 52 and 27% of the total radioactivity recovered by HPLC in dog and human, respectively. When 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, or N-hydroxy-N-acetyl-4-aminobiphenyl was added to human microsomes containing [14C]UDP-glucuronic acid, a new product peak was detected by HPLC. At 0.5 mM, the rate of glucuronidation was N-hydroxy-N-acetyl-4-aminobiphenyl > N-hydroxy-4-aminobiphenyl > 4-aminobiphenyl. The rate of formation of the N-glucuronide of 4-aminobiphenyl was similar to that observed with benzidine and N-acetylbenzidine. The glucuronides of 4-aminobiphenyl and N-hydroxy-4-aminobiphenyl were both acid labile with T1/2 values of 10.5 and 32 min, respectively, at pH 5.5. The glucuronide of N-hydroxy-N-acetyl-4-aminobiphenyl was not acid labile with T1/2 values at pH 5.5 and 7.4 of 55 and 68 min, respectively. The glucuronide of 4-aminobiphenyl was the most acid labile conjugate examined. Thus, the glucuronide of 4-aminobiphenyl is a major product of dog and human liver slice metabolism and likely to play an important role in the carcinogenic process.

Metabolism of N-acetylbenzidine and initiation of bladder cancer

A 100-fold increased incidence of bladder cancer is observed with workers exposed to high levels of benzidine (BZ). This review evaluates the overall metabolism of BZ to determine pathways involved in initiation of carcinogenesis. Enzymatic and liver slice incubations demonstrated N-acetylation and N-glucuronidation of BZ and N-acetylbenzidine (ABZ). With rat, N,N-diacetylbenzidine (DABZ) is the major slice metabolite. With human, ABZ is the major metabolite along with N-glucuronides. Differences between rat and human are attributed to preferential acetylation of BZ and deacetylation of DABZ, resulting in N-glucuronide formation by human liver. Glucuronidation of BZ and its analogues exhibited the following relative ranking of UDP-glucuronosyltransferase (UGT) metabolism: UGT1A9>UGT1A4>>UGT2B7>UGT1A6 approximately UGT1A1. N-Glucuronides of BZ, ABZ, and N-hydroxy-N-acetylbenzidine (NHA) are acid labile with the latter having a much longer t(1/2) than the former two glucuronides. O-Glucuronides are not acid labile. In urine from BZ-exposed workers, an inverse relationship between urine pH and levels of free (unconjugated) BZ and ABZ is observed. This is consistent with the presence of labile urinary N-glucuronides. Cytochrome P-450 oxidizes BZ to an inactive product (3-OHz.sbnd;BZ) and ABZ to NHA and N-hydroxy-N-acetylbenzidine (NHA). Cytochrome P-450, PHS, and horseradish peroxidase activate ABZ to bind DNA forming N-(3-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ). This is the major adduct detected in bladder cells from workers exposed to BZ. An inverse relationship exists between urine pH and levels of bladder cell dGp-ABZ. Bladder epithelium contains relatively high levels of prostaglandin H synthase (PHS) and low levels of cytochrome p-450, suggesting activation by PHS. Activation by PHS involves a peroxygenase oxidation of ABZ to NHA, while horseradish peroxidase activates ABZ to a diimine monocation. Reactive nitrogen oxygen species (RNOS) offer a new pathway for metabolism and potential activation. Results suggest BZ initiation of bladder cancer is complex, involving multiple organs (i.e. liver, kidney, and bladder) and metabolic pathways (i.e. N-acetylation, N-glucuronidation, peroxidation, and RNOS).

Metabolism and action of the prostaglandin endoperoxide PGH2 in rat kidney.

Abstract Kidney membrane fractions metabolized [1-14C]PGH2 to TXB2, PGE2, PGF2α, PGD2, 6-keto PGF1α, and HHT. TXA2, as measured by TXB2, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH2 caused a labile inhibition of cortical PGE2-stimulated adenylate cyclase. PGE2, PGF2α, and PGD2 are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH2 inhibition suggested that TXA2 was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH2 is inhibition of adenylate cyclase.

Effect of dietary calcium and phosphorus restriction on calcium and phosphorus balance in young and old rats.

Abstract Previous studies have shown that the serum levels of the primary regulators of calcium (Ca) and phosphorus (P) metabolism, 1,25-dihydroxyvitamin D and parathyroid hormone, may change with age. Therefore, the effect of age on the ability of the rat to maintain a positive Ca and P balance was determined. Young (1.5 months) and old (18 months) rats were divided into three groups and fed either a low-Ca, high-P diet; a high-Ca, low-P diet; or a high-Ca, high-P diet. After 14 days, the young rats were in positive Ca and P balance regardless of diet. The old rats on the low-Ca, high-P diet were in negative Ca balance and positive P balance. The old rats on the other diets were in positive Ca and P balance. The negative Ca balance of the old rats was due to decreased intestinal absorption of Ca. Intestinal absorption was assessed by determining the percentage of dietary Ca absorbed in vivo and by measuring the active transport of Ca using the everted gut sac in vitro . Intestinal P absorption showed little change with age, except for a decrease in old rats on the high-Ca, low-P diet. Renal adaptation to dietary Ca and P restriction was similar in both young and old animals. Plasma Ca levels were unchanged with age, but plasma P levels decreased with age regardless of diet. These changes in Ca balance with age may reflect the reported decrease in serum 1,25-dihydroxyvitamin D 3 levels and the slight increase in PTH levels with age. The inability of old rats to maintain a positive Ca balance in the face of Ca deprivation is consistent with a general characteristic of the aging process—the decreased ability of an organism to adapt to changes in the external environment.

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Abstract Effects of acetaminophen on the renal inner medullary production of prostaglandin E 2 and F 2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E 2 and F 2α accumulation in media with a K i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E 2 observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E 2 and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E 2 and prostaglandin F 2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.

Enzyme systems involved in the formation of reactive metabolites in the renal medulla: Cooxidation via prostaglandin H synthase☆

Metabolism of drugs and xenobiotics by renal mixed-function oxidases and prostaglandin H synthase was examined. Significant mixed-function oxidase activity was observed in the cortex and outer medulla. However, mixed-function oxidase activity was not detected in the inner medulla. In contrast, prostaglandin H synthase is quite active in the inner and outer medulla with no detectable activity in the cortex. Prostaglandin H synthase was shown to activate a variety of protoxins and procarcinogens by way of its hydroperoxidase activity. Peroxidatic activation of acetaminophen and benzidine appears to involve the formation of a free radical intermediate which binds nucleophilic sites on macromolecules. The latter is proposed to initiate pathogenic effects. Prostaglandin H synthase is a potential alternative to mixed-function oxidase activation of chemicals which exert pathologic effects on the renal inner medulla.

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems☆

Prostacyclin (Prostaglandin I2) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I2 and prostaglandin E2, from 8 · 10−4 to 8 · −7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I2 was more effective than prostaglandin E2 at all concentrations tested. Both prostaglandin I2 and prostaglandin E2 were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I2 and prostaglandin E2 were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I2 stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I2 and prostaglandin E2 (1.5 · 10−4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I2 and prostaglandin E2 increased cyclic AMP content. 6-Ketoprostaglandin F1α, the stable metabolite of prostaglandin I2, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I2 activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I2 may be mediated through the adenylate cyclase-cyclic AMP system.

Peroxygenase Metabolism of N-Acetylbenzidine by Prostaglandin H Synthase FORMATION OF AN N-HYDROXYLAMINE

Synthesis of prostaglandin H2by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mm ABZ to its final end product, 4′-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mm ascorbic acid was present. Similar results were observed whether 0.2 mm arachidonic acid or 0.5 mmH2O2 was used as the substrate. This metabolite co-eluted with syntheticN′-hydroxy-N-acetylbenzidine (N′HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N′HA by electrospray ionization/MS/MS. N′HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N′HA was substituted for ABZ, PHS metabolized N′HA to 4′-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4′-nitro-4-acetylaminobiphenyl, but not for N′HA. A 97% enrichment was observed for one atom of 18O, and a 17 ± 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N′HA with water was suggested to explain the lack of enrichment of N′HA and the low enrichment of two18O atoms into 4′-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N′HA by PHS and suggest a stepwise oxidation of ABZ to N′-hydroxy, 4′-nitroso, and 4′-nitro products.

Differential response of flat and polypoid colitis-associated colorectal neoplasias to chemopreventive agents and heterocyclic amines

Individuals with ulcerative colitis face an increased risk of developing colorectal cancer and would benefit from early chemopreventive intervention. Results from preclinical studies in the mouse model of dextran sulfate sodium-induced colitis demonstrate that flat and polypoid colitis-associated dysplasias arise via distinct genetic pathways, impacted by the allelic status of p53. Furthermore, flat and polypoid dysplasias vary in their response to induction by the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and inhibition by 5-aminosalicylic acid, a common therapy for the maintenance of colitis patients. These data suggest that use of combination therapy is essential for the optimal inhibition of colitis-associated colorectal cancer.

Effects of alamethicin on hormonal activation of renal adenylate cyclase

Abstract The effects of the peptide antibiotic, alamethicin, on hormonally stimulated adenylate cyclase were studied in the rat kidney. In the medullary 100,000 g fraction, antidiuretic hormone-stimulated adenylate cyclase activity was observed only in the presence of alamethicin. Alamethicin augmented the stimulatory effect of parathyroid hormone in the cortical 100,000 g fraction. Lubrol PX solubilized adenylate cyclase activity but, in contrast to alamethicin, did not increase PTH stimulation of adenylate cyclase. Alamethicin had little effect in 1,000 g fractions. Therefore, the effects of this antibiotic do not appear to be due to direct stimulation of adenylate cyclase. In addition, the results cannot be explained by inhibition of phosphodiesterase by alamethicin. Alamethicin is known to increase ionic conductance in membranes by the formation of channels. The effects of alamethicin on adenylate cyclase in the 100,000 g fraction could be due to the formation of channels which increase the permeability of vesicles and thereby increase accessibility to substrate and/or hormones. Different physiochemical properties of renal cortical and medullary plasma membranes are suggested by differences observed in the responsiveness of adenylate cyclase to hormones and to alamethicin. The presence of latent antidiuretic hormone-stimulated adenylate cyclase was observed in the outer medullary 100,000 g fraction.