Reactive nitrogen oxygen species metabolize N-acetylbenzidine.

A close association has been reported for certain types of cancers influenced by aromatic amines and infection/inflammation. Reactive nitric oxygen species (RNOS), components of the inflammatory response, are bactericidal and tumoricidal, and contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible transformation of the aromatic amine N-acetylbenzidine (ABZ) by RNOS. RNOS were generated by various conditions to react with ABZ, and samples were evaluated by HPLC. Conditions which generate nitrogen dioxide radical (NO(2)(-) + myeloperoxidase + H(2)O(2), ONOO(-), and NO(2)(-) + HOCl) produced primarily a single new product termed 3'-nitro-ABZ. The myeloperoxidase-catalyzed reaction with 0.3 mM NO(2)(-) was completely inhibited by 1 mM cyanide, and not effected by 100 mM chloride with or without 1 mM taurine. In contrast, conditions which generate N(2)O(3), such as spermine NONOate, did not produce 3'-nitro-ABZ, but rather two compounds termed 4'-OH-AABP and AABP. (1)H NMR and mass spectrometry identified 3'-nitro-ABZ as 3'-nitro-N-acetylbenzidine, 4'-OH-AABP as 4'-OH-4-acetylaminobiphenyl, and AABP as 4-acetylaminobiphenyl. Human polymorphonuclear neutrophils incubated with [(3)H]ABZ and stimulated with beta-phorbol 12-myristate 13-acetate produced 3'-nitro-ABZ in the presence of NO(2)(-) (0.1-1 mM). Neutrophil 3'-nitro-ABZ formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). The results demonstrate that ABZ forms unique products in the presence of nitrosating and nitrating RNOS, which could influence the carcinogenic process and serve as biomarkers for these reactive species.

Methemoglobin oxidation of N-acetylbenzidine to form a sulfinamide.

Aromatic amine sulfinamide adducts of hemoglobin are biomarkers of exposure and evidence for cytochrome P-450 N-hydroxylation. The possible peroxidatic formation of an N-acetylbenzidine (ABZ) sulfinamide adduct by methemoglobin was examined. Following addition of H2O2, 0.06 mM [3H]ABZ was metabolized by methemoglobin. With 0.3 mM glutathione, a new peak was observed, ABZ-SG, representing 17% of the total radioactivity. N'-Hydroxy-N-acetylbenzidine and 4'-nitro-4-acetylaminobiphenyl were not detected. Optimal ABZ-SG formation was observed with 3 uM methemoglobin, 0.1 to 0.3 mM glutathione, and pH 5.5. Higher concentrations of glutathione were inhibitory. Without glutathione, an H2O2-to-ABZ molar ratio of 1:1 resulted in complete metabolism of ABZ. This ratio increased to greater than 2:1 with 0.3 mM glutathione. Nearly complete inhibition of ABZ-SG formation by cyanide (10 mM), ascorbic acid (0.1 mM), 5,5-dimethyl-1-pyrroline N-oxide (50 mM), thiourea (1 mM), and azide (0.3 mM), and the lack of inhibition by mannitol (50 mM) and superoxide dismutase (2 microg) is consistent with a methemoglobin-mediated peroxidatic reaction, which does not involve hydroxyl radical or superoxide. ABZ-SG was identified by electrospray ionization/mass spectrometry as N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. Conjugate was hydrolyzed by 0.1 N HCl and NaOH, was relatively stable at pH 5.5 and 7.4, and was susceptible to gamma-glutamyltranspeptidase treatment. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The results demonstrate that methemoglobin can catalyze the peroxidatic formation of an ABZ sulfinamide adduct, perhaps by a diimine monocation intermediate.

Nitrating reactive nitric oxygen species transform acetaminophen to 3-nitroacetaminophen.

Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [(14)C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO(2)(-), NO(2)Cl, and ONOO(-) or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO(-), spermine NONO, Sin-1, or H(2)O(2). HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO(2)(-). ONOO(-)-derived 3NAP was identified by (1)H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [(14)C]APAP and stimulated with beta-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO(2)(-). Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO(2)(-). Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.

N-Acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils.

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [(3)H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H(2)O(2), but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P(1) hydrolysis to dG-ABZ, and in (32)P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [(3)H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myristate 13-acetate (PMA). Using (32)P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.

Risk factors for surgical wound infection and bacteraemia following coronary artery bypass surgery.

BACKGROUND: There has been no consensus from previous studies of risk factors for surgical wound infections (SWI) and postoperative bacteraemia for patients undergoing coronary artery bypass graft (CABG) surgery. METHODS: Data on 15 potential risk factors were prospectively collected on all patients undergoing CABG surgery during a 12-month period. RESULTS: Of 693 patients, 62 developed 65 SWI using the Centres for Disease Control definition: 23 were sternal wound infections and 42 were arm or leg wound infections at the site of conduit harvest. There were 19 episodes of postoperative bacteraemia. Multivariate analysis revealed that: (i) diabetes, obesity and previous cardiovascular procedure were independent predictors of SWI; and (ii) obesity was an independent risk factor for postoperative bacteraemia. CONCLUSIONS: These findings suggest that improved diabetic control and pre-operative weight reduction may result in a decrease in the incidence of SWI. But further prospective studies need to be undertaken to examine (i) whether the increased SWI risk in diabetes occurs with both insulin- and non-insulin-requiring diabetes, and whether improved peri-operative diabetes control decreases SWI; and (ii) what degree of obesity confers a risk of SWI and postoperative bacteraemia, and whether pre-operative weight reduction, if a realistic strategy in this patient group, results in a decrease in SWI.

Hypochlorous acid-mediated activation of N-acetylbenzidine to form N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine.

Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important component of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by HOCI to form a specific DNA adduct, N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. HOCl was incubated with 0.06 mM 3H-ABZ, and transformation assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A linear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCI oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected. Oxidation of ABZ was prevented by taurine, DMPO, glutathione, and ascorbic acid, whereas mannitol was without effect. Results are consistent with a radical mechanism. In the presence of 2'-deoxyguanosine 3'-monophosphate (dGp), a new product (dGp-ABZ) was observed. The same adduct was observed with DNA. dGp-ABZ was found to be quite stable (>80% remaining) at 70 degrees C in pH 5.5 (60 min) and 7.4 (240 min). Electrospray mass spectrometry indicated that dGp-ABZ was N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. 32P-postlabeling in combination with TLC and HPLC determined that the adduct made by either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCI activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood cells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.

Sulfinamide formation following peroxidatic metabolism of N-acetylbenzidine.

Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N-oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [(3)H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H(2)O(2) to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to gamma-glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN(3), 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 microgram of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide.

Outcomes of coronary artery bypass grafting: a 3 year analysis using the Society of Thoracic Surgeons Database.

BACKGROUND: Accurate risk factor analysis is a critical element in contemporary cardiac surgical practice. In the USA, the Society of Thoracic Surgeons Database allows institutions and individual surgeons to carry out detailed patient risk assessment and to review their cardiac surgical outcomes in a comparative fashion. METHODS: To evaluate outcomes of isolated coronary artery bypass grafting, data from all patients operated upon at the Alfred Hospital, Melbourne, Australia, over a 3 year period were entered into the Society of Thoracic Surgeons Database. RESULTS: Our results (mortality and morbidity) compared favourably with those contained within this large international database. CONCLUSION: It is hoped that a similar Australasian database can be established to facilitate a meaningful local risk assessment and a comparative analysis of outcomes of cardiac surgical procedures.

Glucuronidation of benzidine and its metabolites by cDNA-expressed human UDP-glucuronosyltransferases and pH stability of glucuronides.

Although glucuronidation is considered a necessary step in aromatic amine-induced bladder cancer, the specific enzymes involved are not known. This study assessed the capacity of five different human recombinant UDP-glucuronosyltransferases expressed in COS-1 cells to glucuronidate benzidine, its metabolites and 4-aminobiphenyl. [(14)C]UDP-glucuronic acid was used as co-substrate. UGT1A1, UGT1A4 and UGT1A9 each metabolized all of the aromatic amines. UGT1A9 exhibited the highest relative rates of metabolism with preference for the two hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidine. UGT1A9 metabolized 4-aminobiphenyl approximately 50% faster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N'-hydroxy- N-acetylbenzidine at the highest relative rate compared with the other transferases. UGT1A6 was effective in metabolizing only four of the eight aromatic amines tested. UGT1A1 demonstrated more extensive metabolism of the hydroxamic acid, N-hydroxy-N,N'-diacetylbenzidine, and the ring oxidation product, 3-OH-N,N'-diacetylbenzidine, than it did for the other six amines. UGT2B7 was the only product of the UGT2 gene family examined and it metabolized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone. The K(m) values for N-acetylbenzidine metabolism by UGT1A1 and UGT1A4 were 0.37 +/- 0.14 and 1.8 +/- 0.4 mM, respectively. The O-glucuronide of 3-OH-N,N'-diacetylbenzidine was not hydrolyzed during a 24 h 37 degrees C incubation at either pH 5. 5 or 7.4. Likewise, the O-glucuronide of 3-OH-benzidine was stable at pH 7.4, with 52% remaining at pH 5.5 after 24 h. These results suggest the following relative ranking of transferase metabolism: UGT1A9 > UGT1A4 > > UGT2B7 > UGT1A6 approximately UGT1A1. The relative pH stability of O-glucuronides is consistent with a role in detoxification and excretion of aromatic amines, while the acid lability of N-glucuronides is consistent with delivery of these amines to the bladder epithelium for activation, resulting in DNA adducts which may lead to mutations.

Human and Escherichia coli beta-glucuronidase hydrolysis of glucuronide conjugates of benzidine and 4-aminobiphenyl, and their hydroxy metabolites.

Individuals exposed to carcinogenic aromatic amines excrete arylamine N- and O-glucuronide metabolites. This study assessed the susceptibility of selected glucuronides to hydrolysis by human and Escherichia coli beta-glucuronidase. N- or O-glucuronides were prepared with the following aglycones: benzidine, N-acetylbenzidine, N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N-hydroxy-N-acetyl-4-aminobiphenyl. The (3)H- and (14)C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 microM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide of N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural isomers. With E. coli beta-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucuronide in the presence of N-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human beta-glucuronidase necessary to achieve significant hydrolysis of glucuronides was substantially more than the amount of enzyme reported previously to be present in urine under either normal or pathological conditions. The bacterial enzyme may hydrolyze O-glucuronides, but not N-glucuronides, in urine at neutral pH. Thus, the nonenzymatic hydrolysis of N-glucuronides by acidic urine is likely a more important source of free amine than enzymatic hydrolysis.

Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. Formation of an N-hydroxylamine.

Synthesis of prostaglandin H2 by 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 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-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 two 18O 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.

Clonidine and cardiac surgery: haemodynamic and metabolic effects, myocardial ischaemia and recovery.

Clonidine may have beneficial effects in patients undergoing major surgery. We enrolled 156 patients having elective CABG surgery in a double-blind, randomized trial. Patients were randomized to receive either two doses of placebo (Group PP) or clonidine 5 micrograms/kg (Group CC). Perioperative measurements included haemodynamics, anaesthetic and analgesic drug usage, creatinine clearance, cortisol excretion, recovery times and quality of life (SF-36) after surgery. Overall, there was no significant difference with time to tracheal extubation (median [10-90 centile]): CC 7.1 (3.4-18) h vs PP 8.0 (4.3-17) h, P = 0.70; but there was a higher proportion of patients extubated within four hours: CC 20% vs. PP 8%, P = 0.038. Clonidine resulted in a number of significant (P < 0.05) haemodynamic changes, particularly pre-CPB: less tachycardia and hypertension, more bradycardia and hypotension. Clonidine was associated with a significant (P < 0.05) reduction in anaesthetic drug usage, higher creatinine clearance, lower cortisol excretion and improvement in some aspects of quality of life. This study lends support to consideration of clonidine therapy in patients undergoing CABG surgery.

N-glucuronidation of benzidine and its metabolites. Role in bladder cancer.

Workers exposed to high levels of benzidine have a 100-fold increased incidence of bladder cancer. This review evaluates the overall metabolism of benzidine to determine pathways important to initiation of bladder cancer. Upon incubation of benzidine with liver slices from rats, dogs, and humans, different proportions of this diamine were N-acetylated and N-glucuronidated. With dogs, a non-acetylator species, N-glucuronidation was the major pathway. In contrast, little glucuronidation was observed in rats with N, N'-diacetylbenzidine, the major metabolite of benzidine. Human liver slices demonstrated both extensive N-acetylation and N-glucuronidation. Differences between rats and humans were attributed to rapid deacetylation by human liver with N-acetylbenzidine rather than an accumulation of N, N'-diacetylbenzidine. N-Acetylbenzidine oxidative metabolism was also observed. The acid lability of glucuronide products of benzidine, N-acetylbenzidine, and oxidation products of N-acetylbenzidine metabolism was assessed. N-Glucuronides of benzidine, N-acetylbenzidine, and N'-hydroxy-N-acetylbenzidine were acid-labile, with the latter having a much longer half-time than the former two glucuronides. Because bladder epithelium contains relatively high levels of prostaglandin H synthase and not cytochrome P450, the peroxidative metabolism of N-acetylbenzidine was assessed. N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was the only DNA adduct detected. This adduct is also the major adduct detected in bladder cells from workers exposed to benzidine. In urine from these workers, an inverse relationship between urine pH and levels of free (unconjugated) benzidine and N-acetylbenzidine was observed. A similar inverse relationship was observed for urine pH and levels of bladder cell N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. These results suggest multiple pathways (acetylation, glucuronidation, peroxidation) in multiple organs (liver, blood, kidney, bladder) are important in benzidine-induced bladder cancer.

N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism.

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced approximately 40-fold more dGp-ABZ than horseradish peroxidase (1943 +/- 339 versus 49 +/- 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.

Urinary mutagenicity as a biomarker in workers exposed to benzidine: correlation with urinary metabolites and urothelial DNA adducts.

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084-5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.

Rat liver cytochrome P450 metabolism of N-acetylbenzidine and N,N'-diacetylbenzidine.

To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism of N-acetylbenzidine and N,N'-diacetylbenzidine was assessed with rat liver microsomes from control and beta-naphthoflavone-treated rats. The oxidation of [3H]N-acetylbenzidine to [3H]N'-hydroxy-N-acetylbenzidine (N'HA), [3H]N-hydroxy-N-acetylbenzidine (NHA), and 3H-ring oxidation products was assessed. For [3H]N,N'-diacetylbenzidine, the formation of [3H]N-hydroxy-N,N'-diacetylbenzidine (NHDA) and the 3H-ring oxidation product was assessed. With beta-naphthoflavone-treated microsomes, the rate of NHA formation was 8-fold more than observed with control. Although significant formation of ring-oxidation products was demonstrated, the formation of N'HA was at the limit of detection. With control microsomes, N'HA was a major metabolite with more N'HA (49 +/- 6 pmol/mg protein/min) produced than NHA (38 +/- 5). Whereas the oxidation of N,N'-diacetylbenzidine was not observed with control microsomes, significant formation of NHDA (421 +/- 49 pmol/mg protein/min) and ring-oxidation (182 +/- 28) product was observed with beta-naphthoflavone-treated microsomes. Metabolism of [3H]N-acetylbenzidine and [3H]N,N'-diacetylbenzidine by beta-naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P4501A1/1A2 inhibitors alpha-naphthoflavone and ellipticine at 10 microM. Except for the < 30% inhibition observed with the cytochrome P4502E1 inhibitor (disulfiram), inhibitors of cytochrome P4503A1/3A2 (troleandomycin) and P4502C6 (sulfinpyrazone) were not effective at 10 microM. N'HA formation by control microsomes was not prevented by any of these inhibitors. Conditions that inhibit flavin-dependent monooxygenase metabolism, methimazole (1 mM), and heat treatment (37 degrees C for 60 min) were also ineffective in preventing N'HA formation. The nonspecific cytochrome P450 inhibitor SKF-525A (10 microM) exhibited a partial dose-response inhibition (maximum 41% of complete reaction mixture) of N'HA formation, but did not alter NHA formation. In contrast, the nonspecific cytochrome P450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine prevented formation of both N'HA and NHA. beta-Naphthoflavone treatment increased [3H]N-acetylbenzidine binding to DNA, but not [3H]N,N'-diacetylbenzidine. Binding of both compounds to DNA was inhibited by ellipticine. N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was detected by 32P-postlabeling in microsomal incubations with N-acetylbenzidine, but not N,N'-diacetylbenzidine. More adduct was detected with control than beta-naphthoflavone-treated microsomes. Results are consistent with cytochrome P4501A1/1A2 playing the major role in N-acetylbenzidine and N,N'-diacetylbenzidine metabolism by liver microsomes from control and beta-naphthoflavone-treated rats. The formation of N'HA by control, but not by beta-naphthoflavone-treated, rats and its insensitivity to inhibition by cytochrome P4501A1/1A2 inhibitors were unexpected.

Hemodynamic effects, myocardial ischemia, and timing of tracheal extubation with propofol-based anesthesia for cardiac surgery.

Recent interest in earlier tracheal extubation after coronary artery bypass graft (CABG) surgery has focused attention on the potential benefits of a propofol-based technique. We randomized 124 patients (34 with poor ventricular function) undergoing CABG surgery to receive either a propofol-based (5 mg.kg-1.h-1 prior to sternotomy, 3 mg.kg-1. h-1 thereafter; n = 58) or enflurane-based (0.2%-1.0%, n = 66) anesthetic. Induction of anesthesia consisted of fentanyl 15 micrograms/kg and midazolam 0.05 mg/kg intravenously in both groups. The enflurane group received an additional bolus of fentanyl 5 micrograms/kg prior to sternotomy and fentanyl 10 micrograms/kg with midazolam 0.1 mg/kg at commencement of cardiopulmonary bypass (CPB). Patients receiving propofol were extubated earlier (median 9.1 h versus 12.3 h, P = 0.006), although there was no difference in time to intensive care unit (ICU) discharge (both 22 h, P = 0.54). Both groups had similar hemodynamic changes throughout (all P > 0.10), as well as metaraminol (P = 0.49) and inotrope requirements (P > 0.10), intraoperative myocardial ischemia (P = 0.12) and perioperative myocardial infarction (P = 0.50). The results of this trial suggest that a propofol-based anesthetic, when compared to an enflurane-based anesthetic requiring additional dosing of fentanyl and midazolam for CPB, can lead to a significant reduction in time to extubation after CABG surgery, without adverse hemodynamic effects, increased risk of myocardial ischemia or infarction.