Lack of methemoglobinemia with flutamide.

OBJECTIVE: To determine whether the nonsteroidal antiandrogenic drug flutamide is a clinically relevant inducer of methemoglobinemia in patients with prostatic cancer. METHODS: Fifty consecutive outpatients with prostatic cancer stage D2 entered the study (age 71.1 +/- 7.3 y). Five patients were lost to follow-up; the remaining 45 patients received the recommended oral dose of flutamide 250 mg three times daily. Total hemoglobin (Hb) and methemoglobin (Met-Hb) concentrations were measured on varying days using an ultraviolet/visible-spectrophotometric method with an intra- and interday variability < 8%. In 12 patients, Met-Hb was analyzed before initiating flutamide therapy and after therapy was begun. RESULTS: On average, 2.6 venous blood samples per patient were analyzed with a mean Met-Hb concentration of 1.9% of total Hb. Mean concentrations of > or = 3% were detected in only six patients (13%). The data from 12 patients evaluated before and after initiating flutamide therapy were without significantly different changes. During the study period, no clinical signs of methemoglobinemia were reported or observed. CONCLUSIONS: This study found no clinically relevant increase of Met-Hb concentrations in elderly patients with prostatic cancer during chronic treatment with flutamide. However, clinicians should be aware of the very rare possibility of flutamide-induced methemoglobinemia.

Direct semiquantitative screening of drugs of abuse in serum and whole blood by means of CEDIA DAU urine immunoassays.

The purpose of this study was to test the direct applicability of CEDIA DAU urine immunoassays to serum or whole blood. The performance of the urine assays for sensitive screening of amphetamines (AMP), benzoylecgonine (BZE), benzodiazepines (BENZ), methadone (MET), opiates (OPI), and tetrahydrocannabinol carboxylic acid (THCCOOH) was evaluated on the BM/Hitachi 911 analyzer with unpretreated serum and whole blood. The limit of detection was 0 ng/mL for all tests. Cutoff values were set from 10 to 40 ng/mL for the different assays. The assays were found to be linear between the following concentrations: AMP 0-2500 ng/mL, BZE 0-1200 ng/mL, BENZ 0-1600 ng/mL, MET 0-600 ng/mL, OPI 0-720 ng/mL, and THCCOOH 24-60 ng/mL. Precision results (within run) for different concentrations were as follows: AMP 3.1-5.7%, BZE 2.4-6.6%, BENZ 4.3-8.0%, MET 2.0-5.5%, OPI 2.8-7.6%, and THCCOOH 1.4-2.4%. Between-run results were as follows: AMP 8.7-15.5%, BZE 6.4-7.5%, BENZ 8.2-15.8%, MET 2.7-5.1%, OPI 4.3-11.2%, and THCCOOH 2.6-7.4%. Sensitivity, specificity, and comparison of CEDIA semiquantitation with GC-MS quantitative results were performed on 500 original serum and whole blood samples. The data provided sufficient documentation to use the CEDIA urine-screening technique without any adaptation as a sensitive serum/whole blood screening for BZE, BENZ, MET, OPI, and THCCOOH. Serum screening for amphetamines is not sensitive enough in the unchanged urine mode. It will require some adaptation to a serum mode (probably a higher sample volume [BM/Hitachi 911] combined with protein precipitation of the sample).

Induction of rat liver microsomal cytochrome P-450 by the pentabromo diphenyl ether Bromkal 70 and half-lives of its components in the adipose tissue.

Pentabromo diphenyl ether (Bromkal 70) was investigated for its half-life in perirenal fat of male and female Wistar rats following a single 300 mg/kg p.o. dose. Half-lives of five individual component fractions following extraction and separation with HPLC were between 30 and 91 days in female rats and between 19 and 119 days in male rats. Induction experiments in male and female Wistar rats with long-term (50 mg/kg daily for 28 days) and short-term administration (300 mg/kg once or 100 mg/kg daily for 4 days) resulted in a 2.3-3.9-fold increase of the cytochrome P-450c, an up to 2-fold increase of benzphetamine N-demethylation activity, a 2.2-5.3-fold increase of the benzo[a]pyrene oxidation activity, and an increase of the ethoxyresorufin O-deethylation activity to between 4.1 and 16.6 nmol/min mg microsomal protein. All variations of enzymatic activities were dependent on the animals' sex or on the administration schedule of the pentabromo diphenyl ether. The threshold for induction of the ethoxyresorufin O-deethylation or benzo[a]pyrene oxidation was between 3 and 10 mg/kg. In conclusion, with regard to the induction of cytochrome P-450 types pentabrominated diphenyl ethers behave as mixed-type inducers.

Monoclonal antibody to the prostate specific antigen.

Somatic cell hybrids were made from mouse myeloma cells and spleen cells derived from BALB/c mice immunized with homogenized epithelial fractions of BPH. The screening by immunoperoxidase staining on human prostate and non-prostate tissue resulted in one monoclonal antibody identifying a prostate specific antigen. Upon SDS-PAGE and Western blot this antigen exhibited a single band at the position of 34 kDa molecular weight. The immunoreactivity of the prostate antigen was found to be localized exclusively in the epithelial lining of ducts and secretions of normal prostate, BPH and prostate cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule and inhibited the binding of Anti-PSA antibody to PSA by about 80 to 90% in the RIA.

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes.

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively. 2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA. 3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.

The pharmacokinetics of flutamide and its major metabolites after a single oral dose and during chronic treatment.

Flutamide is a nonsteroidal antiandrogen used in the treatment of prostatic carcinoma. We have investigated the disposition of flutamide and its two major metabolites in ten urological in-patients without significant liver or renal disease. After oral administration flutamide is absorbed from the gastrointestinal tract with a tmax of about 2 h. Flutamide undergoes extensive first-pass metabolism, and its major metabolites are 2-hydroxyflutamide and the hydrolysis product 3-trifluoromethyl-4-nitroaniline. After the oral administration of a single dose of 250 mg or 500 mg maximum flutamide plasma concentrations of 0.02 and 0.1 micrograms.ml-1 respectively were observed. Maximum plasma concentrations of 2-hydroxyflutamide for the same flutamide doses were 1.3 and 2.4 micrograms.ml-1 (mean of n = 2 or n = 3). Steady-state concentrations of the biologically active metabolite 2-hydroxyflutamide (0.94 +/- 0.23 micrograms.ml-1, mean +/- SD, n = 5) were found at 2-4 days after the administration of 250 mg every 8 h. The area under the plasma concentration time curve for 2-hydroxyflutamide averaged 11.4 (10.6 and 12.1) and 24.3 (21.5-29.4, n = 3) micrograms.ml-1.h for 250 mg and 500 mg flutamide orally. 2-Hydroxyflutamide and 3-trifluoromethyl-4-nitroaniline were eliminated monoexponentially with half-times of 4.3-21.9 and 4.3-17.2 h (n = 5) respectively.

Metabolism of benz(a)anthracene catalyzed by liver microsomes of untreated and nicotine-pretreated rats.

The purpose of the present investigation was to determine whether nicotine causes an induction or alteration of rat liver microsomal monooxygenases (cytochromes P-450) involved in the metabolism and activation of polycyclic aromatic hydrocarbons. After 2, 4, and 10 days of continuous nicotine treatment (16.8 mg/day) neither an increase of benz(a)anthracene metabolism nor an alteration of the metabolite pattern could be observed in comparison to controls. Moreover, there was no significant change of cytochrome P-450 content or benzo(a)pyrene hydroxylation rates. In further control experiments the inducibility of the rats was tested by pretreatment with benzo(k)fluoranthene. As expected, this treatment resulted in a high increase of metabolism and the formation of the ultimate carcinogen as well as other metabolites of benz(a)anthracene due to the induction corresponding to isocytochrome P-450. For nicotine it can be concluded that this alkaloid cannot cause qualitative or quantitative changes of normal rat liver cytochromes P-450.

Monooxygenase induction by various xenobiotics and its influence on rat liver microsomal metabolism of chrysene in comparison to benz[a]anthracene.

The potencies of various xenobiotics for induction of monooxygenases and their influence on the rat liver microsomal metabolite profile of the environmentally relevant weak carcinogen, chrysene, was determined. Among the widely distributed chemicals, polychlorinated biphenyls (PCB) and preferentially 3,3',4,4'-tetrachlorobiphenyl as well as PAHs and their heterocyclic analogues such as benzo[a]pyrene, benzo[b]- and -[j]fluoranthene, indeno[1,2,3-cd]pyrene, dibenz[a,h]acridine, benzo[b]naphtho-[2,1-d]thiophene, and 5,6-benzoflavone were found to be potent inducers stimulating the formation of the proximate, and some of them also the ultimate carcinogen of chrysene. Lindane, carbaryl, DDT, and pentachlorophenol were found to be inefficient or weak inducers. With the exception of phenobarbital no inducers were found among the pharmaceuticals investigated. Sex-dependent metabolism was found for Wistar-rats. No 1,2-oxidation was observed in females, and turnover rates were lower than in males. These findings confirm the results previously obtained with benz[a]anthracene as substrate. The inducing potencies of various compounds tested were similar for both of these substrates. It is interesting to note that in most cases the same effective xenobiotic induces the bay-region diolepoxide in both, chrysene and benz[a]anthracene.

The predominant role of S-oxidation in rat liver metabolism of thiaarenes.

Thiaarenes are metabolized by liver microsomes of untreated rats predominantly to sulfones and sulfoxides. After pretreatment of rats with monooxygenase inducers, ring oxidation of thiaarenes is also observed. In case of benzo[b]naphtho[2,3-d]thiophene the formation of a p-quinone takes place. Rat liver microsomal metabolism of the thiaarenes tested as substrates did not resemble that of the polycyclic aromatic hydrocarbon (PAH) isosters at all.

Monooxygenase induction by various xenobiotics and its influence on the rat liver microsomal metabolite profile of benz[a]anthracene.

Several pesticides (lindane, carbaryl, pentachlorophenol, DDT), polycyclic aromatic hydrocarbons (PAH) and heterocyclic analogues (fluoranthene, dibenz[a,h]anthracene, dibenz[a,h]acridine, indeno[1,2,3-cd]pyrene, 10-azabenzo[a]pyrene) and pharmaceuticals (diphenylhydantoin, ethinylestradiol, levonorgestrel) were tested for their potencies to induce monooxygenase activities in the rat liver by means of recording the metabolite profile of benz[a]anthracene in rat liver microsomal incubations. Some of them were found to be weak or moderate inducers, but even less efficient ones altered the benz[a]anthracene metabolite profile significantly. Only indeno [1,2,3-cd]pyrene stimulated the bay-region oxidation of benz[a]anthracene. A sex-dependent metabolism was observed in both untreated and contraceptive-pretreated Wistar rats.

Induction of specific monooxygenases by isosteric heterocyclic compounds of benz[a]anthracene, benzo[c]phenanthrene and chrysene.

Benzo[c]phenanthrene and a series of heterocyclic compounds (benzo[b]naphtho(1,2-d)thiophene; benzo[b]naphtho(2,1-d)thiophene; benz[a]acridine and benz[c]acridine) were tested to their capacity of inducing monooxygenase activity in rat liver by means of recording the metabolite profile of benz[a]anthracene formed in rat liver microsomal incubations. Although all compounds tested were found to be weak monooxygenase inducers the pretreatment of rats with them resulted in significant changes of the microsomal metabolite profile of benz[a]anthracene. The thiophenes equally gave rise to oxidation at the 5,6- and the 8,9-positions, whereas the benzacridines being isosteric to benz[a]anthracene favoured the K-region oxidation (5,6-oxidation). A structure-dependent effect of monooxygenase inducers on the metabolite profile of benz[a]anthracene is discussed.

Benzo[e]pyrene metabolism in rat liver microsomes: dependence of the metabolite profile on the pretreatment of rats with various monooxygenase inducers.

Benzo[r]pyrene (B[e]P) is metabolized by liver microsomes of untreated rats to trans-4,5-dihydroxy-4,5-dihydrobenzo(e)pyrene and 1- as well as 3-hydroxybenzo[e]pyrene as shown by g.l.c. and mass spectrometry of their trimethylsilyl ethers. After pretreatment of the rats with various monooxygenase inducers oxidation at the 9/10-position was also observed. In addition, secondary oxidation to dihydrodiolepoxides and the formation of a tetrol, tentatively identified as 4,5,9,10-tetrahydroxy-4,5,9,10-tetrahydrobenzo[e]pyrene was detected after incubation of B[e]P with liver microsomes of rats treated with polycyclic aromatic hydrocarbons. However, no formation of the supposed ultimate carcinogen, the 9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene could be observed after any of the pretreatments.

On the metabolic activation of benz[a]acridine and benz[c]acridine by rat liver and lung microsomes.

The metabolism of benz[a]- and benz[c]acridine by liver and lung microsomes from untreated, phenobarbital (PB)-treated and benzo[k]fluoranthene (BkF)-treated rats has been studied by gas chromatography/mass spectrometry (GC/MS). Epoxidation and hydrolysis of the epoxides to dihydrodiols were found to be the predominant pathways for all substrates. N-Oxidation is likely to occur in the case of benz[c]acridine. However, no unequivocal evidence could be obtained for the formation of the ultimate carcinogens--the t-3,4-dihydrodiol-1,2-epoxides--in case of both benz[a]- and benz[c]acridine. K-Region oxidation was induced by phenobarbital, whereas the formation of non-K-region metabolites increased after BkF treatment in the case of benz[c]acridine.

Influence of monooxygenase inducers on the metabolic profile of phenanthrene in rat liver microsomes.

The oxidation of phenanthrene by rat liver microsomes significantly depends on the pretreatment of the animals as shown by means of gas chromatography/mass spectrometry. Whereas untreated animals convert phenanthrene exclusively into the 9,10-dihydrodiol (K-region), pretreatment with various polycyclic aromatic hydrocarbons and related compounds resulted in different rates of additional oxidation at the 1,2- and 3,4-position. Moreover, secondary metabolism to dihydrodiol epoxides, detected as triols, was observed. Despite considerable concentration of the proximate carcinogen of phenanthrene (1,2-dihydrodiol) only low concentrations of the ultimate carcinogen were detected which may explain the carcinogenic inefficiency of this hydrocarbon.

The influence of polycyclic aromatic hydrocarbons as inducers of monooxygenases on the metabolite profile of benz[a]anthracene in rat liver microsomes.

Microsomal oxidation of benz[a]anthracene (BaA) in rat liver has been shown to occur at various positions (1,2-, 3,4-, 5,6-, 8,9- and 10,11-position) by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. In normal rats trans-5,6-, 8,9- and, mainly, 10,11-dihydrodiols have been detected as primary metabolites. The induction of monooxygenases by polycyclic aromatic hydrocarbon (PAH) results in a considerable change in the metabolite profile, since the 5,6- and 8,9-isomers become the main metabolites while the amount of 10,11-isomer is not increased. Simultaneously, the secondary metabolism to form triols and tetrols is induced. Phenobarbital as well as 'moderately inducing' PAH (pyrene, benzo[ghi]perylene, benzo[e]pyrene) induced the oxidation at 5,6- and 8,9-position, whereas almost all other compounds investigated, especially the benzofluoranthenes, additionally induced the oxidation at the 3,4-position forming the precursor of the ultimate carcinogen of BaA, 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene, which was detected as its isomerisation product, the 2,3,4-triol.

Dose-dependent induction of rat liver microsomal aryl hydrocarbon monooxygenase by benzo[k]fluoranthene.

The environmentally widespread polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene is a potent AHH inducer. This has been proven by recording the benz[a]anthracene metabolite profile in the rat liver by means of gas chromatography/mass spectrometry (GC/MS) technique. Even a total dose of 3 times 50 micrograms/kg body wt increases the metabolism of benz[a]anthracene by a factor of 2. The formation of the 8,9- as well as the 5,6-dihydrodiol is stimulated to about the same extent, whereas the formation of the 10,11-dihydrodiol is suppressed. After comparatively low doses of the inducer, a metabolite is formed which corresponds in all parameters with the postulated ultimate carcinogen 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene. This metabolite and a number of other primary and secondary oxidation products could be identified after incubation with induced but not with normal microsomes. Therefore, it should be emphasised that metabolite profiles have to be recorded instead of measuring brutto conversions of PAH substrates to evaluate inducing effects.

Glass-capillary-gas chromatography/mass spectrometry data of mono- and polyhydroxylated benz[a]anthracene. Comparison with benz[a]anthracene metabolites from rat liver microsomes.

By means of glass-capillary-gas chromatography all possible benz[a]anthracene metabolites formed by rat liver microsomes (phenols, dihydrodiols, dihydrodiol enols and tetrahydrotetrols) can be separated. Mass spectra of their trimethylsilyl ethers show intense molecule ions and, in most cases, characteristic fragments. K-Region diols and their secondary oxidation products can be recognized by the ratio (m/e 147) (m/e 191) greater than 1, whereas the ratio is inverse in all other dihydrodiol trimethylsilyl ethers investigated. With the exception of 1,2-dihydrobenz[a]anthracene-1,2,3-triol all vicinal dihydrodiol enols investigated exhibit an intense elimination of the fragment CH = CH-OSiMe3 according to m/e 379. The conformation of vicinal tetrahydrobenz[a]anthracenetetrols possibly can be distinguished by the intensity of m/e 380 (M - 240) since only in those possessing two or more subsequent Me3SiO groups in the same conformation intense elimination of Me3Si-O-CH = CH-O-SiMe3 is observed. Retention times and mass spectrometric data of a series of synthetic benz[a]anthracene derivatives are presented as a base for the identification of benz[a]anthracene metabolites in biological systems.

Time course of oxidative benz[a]anthracene metabolism by liver microsomes of normal and PCB-treated rats.

The time course for the oxidative metabolism of benz[a]anthracene by liver microsomes of normal, 3,3',4,4'-tetrachlorobiphenyl-(TCBP) and polychlorinated biphenyl-(PCB) treated rats has been investigated. These are shown not to be linear in all cases. In normal microsomes the 10,11-dihydrodiol is the main metabolite, followed by the 5,6- and 8,9-dihydrodiols. Secondary metabolism, i.e. formation of dihydrodiol epoxides, is observed only after 5 min. In contrast, TCBP microsomes produced predominantly the 5,6-dihydrodiol followed by the 8,9-dihydrodiol, whereas the formation of the 10,11-dihydrodiol is suppressed. Metabolism deriving from oxidation of the 5,6-position is increased 15-20 fold; again secondary metabolites occur between the 5th and 10th min of incubation. Gas chromatography and mass spectra data suggests the formation of the ultimate carcinogen, 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene, as concluded from detection of its rearrangement product, the 2,3,4-triol. In PCB-treated rats secondary metabolism is observed within 2.5 min. 5,6-Oxidation is increased 27 fold, 8,9-oxidation 10 fold, but 10,11-oxidation is completely suppressed. The above-mentioned ultimate carcinogen is also formed. Moreover, a series of tetrols is detected. Optimum incubation times dependent on the problem under study are discussed.

Studies on the mechanism of stimulation of microsomal H2O2 formation and benzo(a)pyrene hydroxylation by substrates and flavone.

The addition of activators like flavone and hexobarbital to hepatic microsomes markedly stimulates H2O2 formation. The similar increase observed with flavone of microsomal hydroxylation of benzo(a)pyrene and its inhibition by catalase and methanol suggests but does not prove a necessary interaction of microsomal H2O2 production with benzo(a)pyrene hydroxylation. Hexobarbital and flavone-stimulated H2O2 formation is optimal at a stoichiometric relationship of these activators and NADPH. This implies either their direct participation as electron donors or their indirect involvement in electron transport by facilitation of stoichiometric substrate cytochrome P-450/NADPH flavoprotein interactions. Steady state kinetics data are consistent with a scheme in which the formation in microsomes of a complex of 1 mole of NADPH with NADPH-cytochrome P-450 reductase and 1 mole hexobarbital with cytochrome P-450 regulates H2O2 formation.

[Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)]. / Metabolitenprofile von polycyclischen aromatischen Kohlenwasserstoffen nach Vorbehandlung mit verschiedenen Induktoren mikrosomaler Monoxygenasen der Rattenleber.

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.