Identification of the major protein adduct formed in rat liver after thioacetamide administration.

The in vivo covalent binding of the hepatocarcinogen thioacetamide to rat liver protein has been examined. Following administration of 3H- or 14C-labeled thioacetamide, the modified amino acids present in the hepatic cytosolic proteins were isolated by enzymatic digestion and ion-exchange chromatography. Approximately 70% of the radioactivity covalently bound to cytosolic protein was recovered in a compound which upon acid hydrolysis yielded lysine and radiolabeled acetate. Additional studies indicated the structure of this adduct was N-epsilon-acetyllysine.

Dose response for DNA alkylation, [3H]thymidine uptake into DNA, and O6-methylguanine-DNA methyltransferase activity in hepatocytes of rats and mice continuously exposed to dimethylnitrosamine.

The objectives of these experiments were to determine N-7-methylguanine (m7Gua) and O6-methylguanine (O6mGua) concentrations in DNA, [3H]thymidine uptake into DNA, and O6mGua-DNA methyltransferase activity in hepatocytes of F-344 rats and C3H and C57BL mice exposed to 0, 10, 30, or 100 ppm dimethylnitrosamine (DMN) ad libitum in their drinking water for 16 days. The 100-ppm DMN exposure regimen was lethal to the C3H mice. Using water consumption and body weight to surface area conversions, these exposures averaged 5, 13, and 27 mg/sq m/day for F-344 rats, 6, 16, and 31 mg/sq m/day for C57BL mice, and 6 and 16 mg/sq m/day for C3H mice. Over a 5-fold range of DMN exposure, m7Gua concentrations in DNA of rat hepatocytes increased 9-fold, while O6mGua concentrations increased only 3-fold. In contrast, while m7Gua increased 4-fold, O6mGua increased 14-fold in both strains of mice. O6mGua-DNA methyltransferase activity in rat hepatocytes was increased to 150% that of control values at the low exposure, and to 200% at the intermediate and high exposures of DMN. Methyltransferase activity in both strains of mice decreased with increasing exposure to DMN, such that C3H hepatocytes had only 59 and 20% as much activity as controls, while C57BL hepatocytes had 68, 38, and 14% as much methyltransferase activity. Relative to controls, the only significant increase in [3H]thymidine uptake into DNA of hepatocytes occurred at 30 ppm DMN in C3H mice. We conclude that under conditions of DMN exposure leading to comparable m7Gua and O6mGua concentrations in DNA, O6mGua-DNA methyltransferase activity is enhanced in F-344 rats, but partially depleted in C57BL and C3H mice.

Relationships between DNA adduct formation and carcinogenesis.

An impressive array of evidence has been obtained during the past decade establishing correlations between specific DNA adducts and carcinogenesis. Many of the studies utilized organ specific differences in carcinogenesis to establish the correlations. More recently, we have investigated similar relationships between target and nontarget cell populations within the liver. Chronic exposure to methylating hepatocarcinogens predominantly induces hemangiosarcomas, whereas exposure to ethylating agents causes hepatocellular carcinomas. This cell specificity in carcinogenesis correlates well with the presence of promutagenic DNA adducts. In the case of methylating agents, the nonparenchymal cells accumulate O6-methylguanine whereas the hepatocytes do not. Exposure to ethylating agents leads to accumulation of O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine in hepatocytes. These differences reflect the ability of the two cell populations to repair O6-alkylguanine and the extent of purine and pyrimidine alkylation with methylating and ethylating agents. Hepatocytes of rats exposed to diethylnitrosamine for 28 days have four to five times more promutagenic DNA adducts (O6-alkyldeoxyguanosine and O4-alkyldeoxythymidine) than hepatocytes of rats exposed to nearly equimolar doses of dimethylhydrazine. Both O6-methylguanine and O4-methyldeoxythymidine are rapidly repaired by rat hepatocytes, while only O6-ethyldeoxyguanosine is rapidly repaired. Studies comparing the relationship between the induction of gamma-glutamyl transpeptidase-positive foci, hepatocellular carcinoma and promutagenic lesions such as O4-ethyldeoxythymidine will be useful in understanding associations between the molecular dosimetry of DNA adducts, initiation, and progression of hepatocarcinogenesis.

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Inhibition of human drug metabolizing cytochromes P450 by anastrozole, a potent and selective inhibitor of aromatase.

Anastrozole (2,2'[5(1H-1,2,4-triazol-1-ylmethyl)-1,3-phenylene]- bis(2-methylproprionitrile)) is a potent third-generation inhibitor of aromatase, currently marketed as a treatment for postmenopausal women with advanced breast cancer. While its potency and selectivity for inhibition of estrogen synthesis has been established in both preclinical and clinical studies, this study used in vitro methods to examine the effects of anastrozole on several drug metabolizing CYP enzymes found in human liver. Human liver microsomes were co-incubated with anastrozole and probe substrates for CYP1A2 (phenacetin), CYP2A6 (coumarin), CYP2C9 (tolbutamide), CYP2D6 (dextromethorphan), and CYP3A (nifedipine). The formation of the CYP-specific metabolites following co-incubation with various anastrozole concentrations was determined to establish IC50 and Ki values for these enzymes. While anastrozole did not inhibit CYP2A6 and CYP2D6 activities at concentrations below 500 microM, this compound inhibited CYP1A2, CYP2C9, and CYP3A activities with Ki values of 8, 10, and 10 microM, respectively. Dixon plots used to determine the Ki values for the inhibition of CYP1A2 and CYP3A activities by anastrozole were biphasic, indicating additional lower affinity Ki values. Major metabolites of anastrozole did not retain the ability to inhibit the metabolism of nifedipine (CYP3A). The results of this study indicate that, although anastrozole can inhibit CYP1A2, 2C9, and 3A-mediated catalytic activities, this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at physiologically relevant concentrations achieved during therapy with Arimidex (Zeneca, Ltd., Macclesfield, UK) 1-mg.

Hepatic macromolecular covalent binding of the hepatocarcinogen 2,6-dinitrotoluene and its 2,4-isomer in vivo: modulation by the sulfotransferase inhibitors pentachlorophenol and 2,6-dichloro-4-nitrophenol.

The sulfotransferase inhibitors 2,6-dichloro-4-nitrophenol and pentachlorophenol were used to investigate the role of sulfate ester formation during the in vivo bioactivation of 2,4- and 2,6-dinitrotoluene (DNT). Male F-344 rats were administered one of the sulfotransferase inhibitors (40 mu mol/kg i.p.) 45 min prior to oral administration of 28 mg/kg [ring-14C]-2,4-DNT or [3-3H]-2,6-DNT and killed 12 h later. Pentachlorophenol had no significant effect on the urinary excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. The sulfotransferase inhibitors decreased the total hepatic macromolecular covalent binding of 2,4-DNT by 33%, and of 2,6-DNT by 69%. Purification of hepatic DNA by hydroxylapatite chromatography indicated covalent binding of 2,4- and 2,6-DNT at levels of 45 and 94 pmol equivalents/mg DNA, respectively. The sulfotransferase inhibitors decreased the binding of the hepatocarcinogen 2,6-DNT to hepatic DNA by greater than 95%. 2,6-Dichloro-4-nitrophenol decreased the binding of 2,4-DNT to DNA by greater than 84% while the decrease due to pentachlorophenol was 33%. These results suggest that sulfation is important in the biotransformation of 2,4- and 2,6-DNT to reactive metabolites which covalently bind to DNA. 3H2O was detected in the urine of rats administered [3-3H]-2,6-DNT. Pentachlorophenol decreased 3H2O formation to the same extent as it decreased the total hepatic macromolecular covalent binding of 2,6-DNT, suggesting that 3H exchange at the 3 position of 2,6-DNT occurs following sulfate ester formation. These results are consistent with a nitrenium-carbonium ion resonance of the sulfate ester-derived reactive intermediate of 2,6-DNT.

Quantitation of etheno adducts by fluorescence detection.

Etheno adducts can be formed by the reaction of vinyl chloride metabolites with DNA and may play a role in the carcinogenicity of this chemical. These adducts are highly fluorescent and may be quantitated by sensitive photometric methods in conjunction with high-performance liquid chromatographic (HPLC) separation. Three HPLC systems were evaluated on the basis of maximal fluorescence intensity and resolution of two etheno adducts, ethenodeoxycytidine and ethenodeoxyadenosine. Analyses were conducted with enzymatically digested DNA that had been incubated with chloroacetaldehyde, a vinyl chloride metabolite which may cause etheno adduct formation in vivo. All three known etheno adducts of DNA were tentatively identified in DNA reacted in vitro. The sensitivity of the method was highest for the ethenodeoxyadenosine adduct, with the limit of detection (1 pmol per injection in the HPLC system) being similar to that for O6-methylguanine, another promutagenic DNA adduct for which quantitation by HPLC with fluorescence detection has been reported. The method described here may be useful for the analysis of DNA from animals or humans exposed to vinyl chloride.

Differential repair of O4-alkylthymidine following exposure to methylating and ethylating hepatocarcinogens.

Recent experiments have demonstrated that O6-alkylguanine is rapidly removed from hepatocyte DNA following continuous exposure to 1,2-dimethylhydrazine or diethylnitrosamine. In contrast, O4-ethyldeoxythymidine accumulates to concentrations more than 50 times greater than O6-ethyldexyguanosine. Studies on the formation and persistence of O4-methyldeoxythymidine in vivo have not been reported. This study reports the development of sensitive radioimmune assays to O4-methyldeoxythymidine and O4-ethyldeoxythymidine. Utilizing this method, the accumulation and removal of O4-methyldeoxythymidine and O4-ethyldeoxythymidine in liver DNA from rats exposed to 1,2-dimethylhydrazine or diethylnitrosamine were measured. The results demonstrated that O4-methyldeoxythymidine was formed at an O6-methylguanine/O4-methyldeoxythymidine ratio of approximately 100/1 and was repaired with a half-time of approximately 20 h. In contrast, O4-ethyldeoxythymidine removal was 13 times slower with a t 1/2 of approximately 11 days after both pulse dose and cessation of continuous DEN administration. Combined with previously reported data, results presented here suggest that (i) despite a lower rate of formation, O4-methyldeoxythymidine becomes nearly equal in importance to O6-methylguanine as a promutagenic adduct in hepatocytes from continuously exposed rats and (ii) differential repair of O4-alkylthymidine adducts provides a mechanism that may explain in part the superior ability of ethylating versus methylating agents to induce hepatocellular carcinomas in the rat.

Catalytic selectivity and mechanism-based inactivation of stably expressed and hepatic cytochromes P450 2B4 and 2B5: implications of the cytochrome P450 2B5 polymorphism.

Cytochrome P450 (P450) 2B5 was recently found to be functionally distinct from three other rabbit P450 2B forms, based on androstenedione hydroxylase activities. In this investigation, we examined the frequency of the P450 2B5-null phenotype and the functional consequences of polymorphic P450 2B5 expression in hepatic microsomes from phenobarbital-treated rabbits. Four of the 10 animals examined did not have detectable levels of P450 2B5 mRNA and exhibited much lower microsomal androstenedione 15 alpha- and 16 alpha-hydroxylase activities. The 15 alpha-hydroxylase activity was found to correlate (r = 0.91) with liver P450 2B5 mRNA. P450 2B4 and 2B5 were stably expressed in human kidney 293 cells to further characterize substrate specificities and to investigate mechanism-based inactivation by phencyclidine. P450 2B4 was 4-16-fold more active than 2B5 towards benzphetamine, 7-ethoxycoumarin, methylenedioxybenzene, and pentoxyresorufin. Benzyloxyresorufin O-debenzylase activity was 160-fold higher for P450 2B4 than P450 2B5. Anti-P450 2B4 IgG inhibited benzyloxyresorufin O-debenzylation nearly completely in untreated and phenobarbital-induced liver microsomes. Phencyclidine selectively inactivated P450 2B4, compared with 2B5, in both human kidney 293 cell and liver microsomes. Poor inactivation of P450 2B5 by phencyclidine was found to be a result of its low maximal rate constant. Results of this study establish the idea that the metabolic consequences of phenobarbital induction depend on the potential of animals to express functionally variant P450 2B forms. Furthermore, we conclude that one or more of the 11 amino acid differences between these highly related P450 forms are critical to their substrate specificities and selective inactivation.

Determination of ZD9583, a thromboxane receptor antagonist, in human plasma and urine by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry.

A liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) method is described for the determination of a thromboxane receptor antagonist (4Z)-6-((2S,4S,5R)-2-(1-(2-cyano-4-methylphenoxy)-1-methylethyl)-4 -(3-pyridyl)-3-dioxan-5-yl)hex-4-enoic acid (ZD9583, I) in human plasma and urine. Proteins in plasma and urine samples are precipitated using acidified acetonitrile. The resulting supernatant is chromatographed on a C8 reversed-phase chromatography column. Following the diversion of the solvent front from the mass spectrometer by a switching valve, the column eluate is passed on to the mass spectrometer via a heated nebulizer interface where the analyte is detected by multiple reaction monitoring (MRM). The method has a chromatographic run time of less than 2 min, a linear calibration curve with a range of 1-500 ng ml(-1) and intra- and inter-day precision estimates of less than 10% over the calibration range.

Correlation of O4-ethyldeoxythymidine accumulation, hepatic initiation and hepatocellular carcinoma induction in rats continuously administered diethylnitrosamine.

Recent experiments have demonstrated that O6-ethyldeoxyguanosine (O6-EtdG) is efficiently repaired while O4-ethyldeoxythymidine (O4-EtdT) accumulates in hepatocyte DNA of 8-week-old F-344 rats during continuous diethylnitrosamine (DEN) administration. To determine if O4-EtdT accumulation correlates with hepatic initiation, we have quantitated O4-EtdT concentrations, and the incidence of gamma-glutamyl transferase positive (GGT+) foci and hepatocellular carcinoma induced by increasing duration of exposure to DEN in the drinking water (40 p.p.m.). In 8-week-old F-344 rats the number of GGT+ foci increased non-linearly with duration of exposure and reached a maximum of approximately 500 foci/cm3 after 10 weeks. Administration of DEN to 8-week-old F-344 rats for 6 weeks followed by a 15-week administration of 0.05% phenobarbital (PB) in the diet did not result in the induction of hepatocellular carcinoma. Exposure of 4-week-old F-344 rats to DEN for up to 10 weeks produced an O4-EtdT steady-state concentration (approximately 7-10 X 10(-6) mol O4-EtdT/mol dT) similar to that previously observed after administration of DEN to 8-week-old F-344 rats. However, the maximal concentration of O4-EtdT was detectable after a shorter period of DEN administration in the younger rats. The incidence of GGT+ foci also increased more rapidly in 4-week-old rats, but again plateaued at approximately 500 foci/cm3 after 4, 6 or 8 weeks of DEN administration. A 100% incidence of hepatocellular carcinoma occurred in 4-week-old rats administered DEN for 6, 8 or 10 weeks, followed by promotion with 0.05% PB in the diet until week 22 of the study. Lower incidences of hepato-cellular carcinoma (89 and 6%) were observed following PB-promotion of rats administered DEN for 4 and 2 weeks, respectively. The influence of age on DEN-induced hepatic initiation was examined further by quantitating GGT+ foci induced by 4 weeks of DEN administration in groups of rats which were 4-14 weeks old at the start of the carcinogen exposure. The results demonstrated that the younger rats were 15-fold more susceptible than the older rats to the initiating effects of DEN. This growth-dependent effect on hepatic initiation in the presence of nearly equivalent amounts of pro-mutagenic DNA damage further implicates the necessity of replication for hepatic initiation.

Biochemical and morphologic studies of heterogeneous lobe responses in hepatocarcinogenesis.

Experiments have demonstrated interlobe differences in the incidence of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCCA), with a 100% incidence in the left and right median lobes and a 30% incidence in the right anterior lobe 20 weeks after exposure began. These tumor data provide a model to test the hypothesis that chemically induced neoplasia can be qualitatively and quantitatively related to promutagenic DNA damage and concurrent cell replication. Experiments were performed to measure O4-ethyldeoxythymidine (O4-EtdT) (a major pro-mutagenic lesion in hepatic DNA of rats exposed to DEN), N7-ethylguanine, cell replication and hepatocyte initiation using the induction of growth-selected gamma-glutamyl transferase-positive (GGT+) foci in the left and right median and right anterior hepatic lobes following 0, 3, 7, 14 or 28 days of DEN administration. Results demonstrated that O4-EtdT concentrations were consistently higher in the left and right median versus the right anterior hepatic lobes, while cell replication was transiently higher in the right median and right anterior lobes. Likewise, high numbers of GGT+ foci were observed in the left and right median lobes in DEN-exposed rats subjected to a Cayama-Farber growth selection protocol. Following administration of [14C]DEN, the distribution of radioactivity showed a marked left lobe preference in 4-week-old rats that had no prior exposure to DEN and in 8-week-old rats exposed to DEN for 4 weeks. This study suggests that interlobe differences in hepatocyte initiation and the incidence of HCCA may be due in part to differences in cell replication and in DNA alkylation resulting from differential DEN distribution and/or metabolism.

O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine.

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.