Metabolism of 3,3'-dichlorobenzidine by horseradish peroxidase

1. The peroxidatic oxidation of 3,3'-dichlorobenzidine by horseradish peroxidase in the presence of H2O2 was examined spectrophotometrically and the reactivity of the spectral species were compared to those formed from the peroxidative oxidation of benzidine. 2. The horseradish peroxidase-catalyzed oxidation of 3,3'-dichlorobenzidine yielded two transient and one stable spectral species with absorption maxima at 630 nm, 370 nm and 410 nm, respectively, whereas that of benzidine yielded three stable spectral species with absorption maxima at 610 nm, 425 nm and 370 nm, respectively. 3. The 425 nm species from benzidine, but not the 410 nm species from 3,3'-dichlorobenzidine was scavenged by butylated hydroxyanisole, glutathione, N-acetylcysteine or 2-deoxyguanosine. 4. H.p.l.c. mass spectrometric analysis, and comparative studies with potassium dichromate oxidation of 3,3'-dichlorobenzidine, indicated that the major product from the horseradish peroxidase-catalyzed oxidation of 3,3'-dichlorobenzidine is azo-3,3'-dichlorobenzidine. 5. None of the products from enzymic or chemical oxidation of either 3,3'-dichlorobenzidine or benzidine was directly mutagenic to S. typhimurium TA98 in the Ames test; however the chemically oxidized and enzymic products from 3,3'-dichlorobenzidine were mutagenic in the presence of H2O2. 6. The data indicate that despite apparent structural similarities between the intermediates formed during the peroxidatic oxidation of all benzidines, the intermediates and products of peroxidatic oxidation of dichlorobenzidine have reactivities and stabilities different from those of other benzidines.     

Covalent interaction of 3,3'-dichlorobenzidine with hepatic lipids. Enzymic basis and stability of the adducts

Administration of a single oral dose (20 mg/kg) of [U-14C]3,3'-dichlorobenzidine to rats resulted in the in vivo covalent binding of the compound to hepatic lipids. More than 70% of the lipid-3,3'-dichlorobenzidine adducts were accounted for in microsomes. Loss of the lipid-bound 3,3'-dichlorobenzidine residues from either total liver or endoplasmic reticulum occurred in at least two phases--an initial fast phase and a terminal slow phase. In vitro studies with hepatic microsomes in the presence of antibodies to specific P450 isozymes and chemical inhibitors to determine the enzymes that activate 3,3'-dichlorobenzidine to the lipid-binding derivative(s) implicated cytochrome P450d. The 3,3'-dichlorobenzidine-bound microsomal lipids were not mutagenic to Salmonella TA98 in the Ames test. The results suggest that adduct formation between 3,3'-dichlorobenzidine and membrane lipids may provide a measure of 3,3'-dichlorobenzidine activation. It is speculated that covalent interaction of the compound with membrane lipids may modify cellular processes, leading to either enhancement or attenuation of carcinogenesis by the chemical.     

The kinetics of 4-nitrophenol conjugation by perfused livers and hepatic microsomes from streptozocin-induced diabetic rats

The formation of both glucuronide and sulphate conjugates of 4-nitrophenol is deficient in perfused livers from male diabetic rats. Experiments with ‘native’ hepatic microsomes demonstrated that the defect in glucuronidation is due to a decrease in the maximal velocity of the reaction. There is no alteration in the affinity of the glucuronyltransferase for 4-nitrophenol. Non-linear regression analysis of the 4-nitrophenol liver perfusate concentrations snowed that the elimination follows saturable Michaelis-Menten kinetics. Clearance values in ‘native’ microsomal preparations and in perfused livers were calculated and found to be similar in both systems. This provides evidence that glucuronyltransferase is ‘native’ in the intact liver.     

Development of a strategy for biological monitoring in a chemical plant producing 3,3'-dichlorobenzidine dihydrochloride

In a chemical plant in Germany producing 3,3'-dichlorobenzidine dihydrochloride for the manufacture of colorants, blood and urine samples were taken for biological monitoring. 3,3'-Dichlorobenzidine (DBZ) was analyzed in urine by thin-layer chromatography and subsequently further combined with analysis of adducts of 3,3'-DBZ in hemoglobin. Data highlight current ranges of industrial exposure to 3,3'-DBZ in Germany and demonstrate the applicability of biological monitoring to minimize this exposure. Effective biological monitoring was achieved by a combination of monitoring hemoglobin adducts with spot samplings of urinary 3,3'-DBZ excretion in cases of reported exposure periods. Data presented might help to identify biological guidance values (BGV/BAR) for 3,3'-DBZ-exposed individuals.     

Development and evaluation of a microtiter plate enzyme immunoassay for antibodies to 3,3'-dichlorobenzidine

The authors obtained and evaluated antisera from rabbits injected with a derivative of a potent bladder carcinogen, dichlorobenzidine (DCB), conjugated to bovine serum albumin (BSA). A 14C-radioimmunoassay (RIA) was able to detect the presence of DCB antibodies, but its relative insensitivity led to the development of a more sensitive enzyme immunoassay (EIA). The EIA test was a "sandwich" method in which a second antibody, labeled with an enzyme (horseradish peroxidase), was used to measure antibody binding to transferrin (Tf)-conjugated DCB immobilized on a microtiter plate. Antibody titers measured by RIA were approximately 1:40; when measured by EIA, they were approximately 1:40,000. Antibody specificity was assessed by comparing the antibody binding activities of DCB, BSA, Tf, BSA-conjugated to DCB, and a number of N-substituted aromatic compounds that included benzidine (Bz). Among the compounds tested, the rabbit antiserum reacted only with DCB and the carrier protein, BSA. Moreover, antibody binding activity to Tf-conjugated DCB was significantly inhibited by unconjugated DCB concentrations between 30 and 500 ng/mL. The precision of antibody binding activities as a function of DCB concentration (expressed by the CV) ranged from 9% for low (30 ng/mL) DCB levels to 12% for higher (500 ng/mL) levels. This evaluation suggests that the antiserum obtained would be appropriate for detecting DCB levels at the ng/mL level.     

甲基丁香酚的大鼠肝微粒体生物转化

甲基丁香酚（1）是中药肉豆蔻的主要成分之-,将其与大鼠肝微粒体共温孵,从温孵物中通过色谱方法得到原形化合物1和8个转化产物（2-9）,根据谱学数据并与文献报道数据比较,确定它们分别为（R）-1-甲氧基甲基丁香酚（2）、3-（3,4-二甲氧基苯基）-1-甲氧基-2-丙烯（3）、顺式-3,4-二甲氧基桂皮酰乙酯（4）、反式-3,4-二甲氧基桂皮酰乙酯（5）、（R）-1-羟基甲基丁香酚（6）、反式-3,4-二甲氧基桂皮醇（7）、顺式-3,4-二甲氧基桂皮醇（8）、僻）-3-（3,4-二甲氧基苯基）-丙烷-1,2。二醇（9）。化合物7和8的13C NMR数据为首次报道。本文结果为甲基丁香酚的应用提供了有用的信息。     

餐时血糖调节剂米格列奈钙的RP-HPLC法人肝微粒体药物浓度测定

目的:建立人肝微粒体中米格列奈的高效液相色谱测定法.方法:采用Diamonsil-C18色谱柱(200 mm×4.6 mm,5μm);流动相为乙腈-0.02 mol·L-1KH2PO4缓冲液(pH 4.0)(43:57);流速为1.0 mL·min-1;紫外检测波长为210 nm;进样量为20 mL.结果:米格列奈最低检测限为2 μmol·L-1;线性范围为5～1 000 μmol·L-1(r=0.999 9);最低定量浓度为5μmol·L-1(RSD<5%,n=5);低、中、高3种浓度日内、日间RSD(n=5)分别为1.9%,1.6%,1.4%和3.8%,4.0%,3.8%;绝对回收率平均为94.4%,相对回收率平均为101.2%.结论:本实验建立的人肝微粒体中米格列奈的高效液相测定方法操作简便、结果准确.     

Metabolism of methyltertiary-butyl ether by rat hepatic microsomes

Exposure to methyltertiary-butyl ether (MTBE), a commonly used octane booster in gasoline, has previously been shown to alter various muscle, kidney, and liver metabolic activities. In the present study, the metabolism of MTBE by liver microsomes from acetoneor phenobarbital-treated Sprague-Dawley rats was studied at concentrations of up to 5 mM MTBE. Equimolar amounts oftertiary-butanol, as measured by head-space gas chromatography, and formaldehyde were formed. TheV _max for the demethylation increased by 4-fold and 5.5-fold after acetone and phenobarbital treatments, respectively. The apparentK _m value of 0.70 mM using control microsomes was decreased slightly after acetone treatment, but was increased by 2-fold after phenobarbital treatment. The metabolism of MTBE (1 mM) was inhibited by 35% by monoclonal antibodies against P450IIE1, the acetone/ethanol inducible form of cytochrome P450, suggesting a partial contribution by this isozyme. A single 18-h pretreatment of rats with 1 or 5 ml/kg MTBE (i. p.) resulted in a 50-fold induction of liver microsomal pentoxyresorufin dealkylase activity but no change inN-nitrosodimethylamine demethylase activity. These trends in activity agreed with immunoblot analysis which showed an elevation in P450IIB1 but no change in P450IIE1 levels.The nomenclature for the P450 isozymes follows the convention described by Nebert et al. (1987).Supported by Grant ES-03938 from the National Institutes of Health and Grant 88B18 from the American Institute for Cancer Research     

Epoxidation of 4-vinylcyclohexene by human hepatic microsomes

Carcinogenicity studies have shown that chronic administration of 4-vinylcyclohexene (VCH) will induce ovarian tumors in B6C3F1 mice but not F-344 rats. This occurs because the blood level of the ovotoxic VCH metabolite, VCH-1,2-epoxide, is dramatically higher in VCH-treated female mice compared with rats. This species difference in VCH epoxidation is also reflected in the rate of VCH metabolism by hepatic microsomes (female mouse greater than female rat). The present study assessed the ability of microsomes obtained from human liver to metabolize VCH to epoxides since humans are exposed to VCH in certain occupational settings. The production of VCH-1,2-epoxide and VCH-7,8-epoxide from VCH (1 mM) by human hepatic microsomes was linear with respect to microsomal protein concentration (0.25-1.0 mg/ml) and incubation time (5-20 min). VCH-1,2-epoxide was the major metabolite, while the rate VCH-7,8-epoxide formation was about 6-fold lower and in some cases was below the limit of detection. There was no dramatic difference in the rate of VCH epoxidation by hepatic microsomes obtained from male and female humans. The rate of VCH-1,2-epoxide formation by female human hepatic microsomes was 0.71 +/- 0.35 nmol/mg microsomal protein/min (n = 4). This is 13- and 2-fold lower than the rate of VCH-1,2-epoxide formation by female mouse and rat hepatic microsomes, respectively. Therefore, if the rate of hepatic VCH epoxidation is the main factor which determines the ovotoxicity of VCH, then the results of these studies suggest that rats are the more appropriate animal model for extrapolation of animal data to humans.     

Characterization of the acetaminophen-glutathione conjugation reaction by liver microsomes: species difference in the effects of acetone

NADPH-dependent production of acetaminophen-glutathione conjugate has been partly characterized in rat liver microsomes. The reaction showed the characteristics of mixed function oxidase when glutathione concentration was higher than 0.2 mM. It is suggested that hydroxy radical and superoxide are not involved in this reaction. When the reaction was compared among rat, mouse and rabbit microsomes, mouse microsomes showed the highest activity; this was almost 4 times higher than the others, which may, at least in part, explain the susceptibility of mouse to acetaminophen-induced hepatotoxicity. There was also a species difference in the effects of acetone; this was enhanced in the rat microsomes, inhibited in the mouse, and minimally affected in the rabbit.     

反相高效液相色谱法测定鼠肝微粒体中依普黄酮及其在代谢研究中的应用

目的　建立鼠肝微粒体中依普黄酮的反相高效液相色谱测定法 ,以研究依普黄酮的体外代谢。方法　本法中依普黄酮与鼠肝微粒体共孵育之后用氯仿提取 ,采用地非三唑为内标 ,以Nova parkC18柱为分析柱 ,乙腈 0 .1%醋酸溶液 ( 6 0∶4 0 )为流动相 ,流速 1.0mL·min-1,紫外检测波长为 2 5 0nm。结果　依普黄酮在 1～ 10 0 μg·mL-1内线性关系良好 (r =0 .9998)。检测限为 0 .0 2 μg·mL-1(S/N≥ 3) ,定量限为 0 .1μg·mL-1(RSD <5 .0 % ,S/N =8,n =3)。方法回收率达 96 .90 %～ 112 .8% ,日内 ,日间RSD分别 <8.0 %和 <10 % (n =5 )。结论　此法简便 ,准确 ,可用于依普黄酮的体外代谢研究     

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein⁻¹ min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.     

Stimulation of calcium uptake of muscle microsomes by phenothiazines and barbiturates

The calcium pump of sacroplasmic reticulum of rabbit and frog is stimulated by phenothiazines in the concentration range 10–100 μM, and by barbiturates in the concentration range 0.5–4.0 mM at 25°C. When potassium, which is an intracellular activator of the calcium pump, is incorporated in the medium in 100 mM concentration the phenothiazines, and less so the barbiturates, reduced the rate of calcium uptake. Chlorpromazine sulphoxide, a pharmacologically inactive metabolite of chlorpromazine, did not activate in the absence of potassium nor inhibit in its presence. Barbituric acid also was without effect. When the “extra” AtPase was measured during chlorpromazine-stimulated calcium uptake no increase in efficiency of the system was detected.     

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.     

Metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl by human hepatic microsomes

Since the metabolism of polychlorinated biphenyls (PCBs) is the critical factor that determines whether or not they accumulate in adipose tissue, we have studied the metabolism of two hexachlorobiphenyls (HCBs), 2,2'3,3',6,6'-hexachlorobiphenyl (236-HCB) and 2,2'4,4',5,5'-hexachlorobiphenyl (245-HCB), by human hepatic microsomes. Human microsomes were isolated from patients undergoing liver resection and were found to have cytochrome P-450 levels (0.28 nmoles/mg microsomal protein) and cytochrome P-450-dependent enzymatic activities similar to those reported by other workers. 245-HCB was not metabolized by human microsomes under various conditions, while 236-HCB was metabolized with an apparent Km of 8.8 microM and a Vmax of 5.1 pmoles/mg microsomal protein/min. Two major metabolites were formed and identified by gas chromatography-mass spectrometry as 2,2',3,3',6,6'-hexachloro-4-biphenylol and 2,2',3,3'6,6'-hexachloro-5-biphenylol. [14C]236-HCB equivalents were found to covalently bind to microsomal protein. Addition of 1 or 5 mM reduced glutathione decreased the degree of covalent binding. These data suggest that HCBs are metabolized through an arene oxide. The fact that 245-HCB was not metabolized explains why it is the predominant PCB found in human adipose tissue.