NADPH-cytochrome P-450 Reductase Activity Research Articles

Inhibition of purified and membrane-bound flavin-containing monooxygenase 1 by (N,N-dimethylamino)stilbene carboxylates.

(E)-[2-(4-(Dimethylamino)phenyl)vinyl]benzenes bearing a nitrile or carboxyl group in the 2', 3', or 4' position were synthesized and tested for substrate activity with purified pig liver flavin-containing monooxygenase (FMO1). Although the nitrile derivatives were too insoluble to saturate the catalytic site at pH 7.4, they appeared to be substrates with K(m)'s somewhat above their maximum solubility (approximately equal to 0.1 mM) in the assay medium. Of the three carboxylic acid analogs, (E)-4-[2-(4(dimethylamino)phenyl)vinyl]benzoic acid had no detectable water solubility at pH 7.4, and measurements were restricted to (E)-3-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS3CO) and (E)-2-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS2CO). While DS3CO and DS2CO were substrates, they also inhibited FMO1 turnover. DS3CO was the more effective inhibitor, and at 2 mM it inhibited FMO1 and microsomal-catalyzed oxidation of methimazole (N-methyl-2-mercaptoimidazole) by 80-90%. Kinetic studies indicated that the aminostilbene carboxylates were noncompetitive with both the xenobiotic substrate, methimazole, and NADPH. However, inhibition constants calculated from double reciprocal plots of velocity vs NADPH were K(i)(comp) 130 and 150 microM for DS3CO and DS2CO, respectively, whereas the uncompetitive Ki's were 10-15 times higher, which suggests that inhibition of NADPH binding may be primarily responsible for inhibition of FMO1 by the aminostilbene carboxylates. This model is also consistent with inhibition of cyclohexanone monooxygenase, a bacterial analog of FMO. DS3CO and DS2CO were again noncompetitive with methimazole but primarily competitive with NADPH. The aminostilbene carboxylates had no detectable effects on activity of pig or rat liver NADPH-cytochrome P450 reductase, which suggests that they are not nonspecific flavoprotein antagonists.

Isoflurane acts as an inhibitor of oxidative dehalogenation while acting as an accelerator of reductive dehalogenation of halothane in guinea pig liver microsomes

The effects of isoflurane, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, on the oxidative metabolism of halothane to produce trifluoroacetic acid (TFA) and on the reductive metabolism of halothane to produce chlorodifluoroethylene (CDE) and chlorotrifluoroethane (CTE) in liver microsomes of guinea pig were examined. Isoflurane enhanced the production of CDE and CTE and inhibited the production of TFA. Isoflurane enhanced cytochrome P450 reduction and formation of an intermediate complex with cytochrome P450 without enhancement of NADPH-cytochrome P450 reductase (EC 1.6.2.4) activity. We conclude that isoflurane interacts with cytochrome P450 to prevent the formation of the halothane-cytochrome P450 complex, causing inhibition of the oxidative dehalogenation. This interaction of isoflurane enhances the reduction of cytochrome P450 and the formation of a reductive intermediate-cytochrome P450 complex under anaerobic conditions causing reductive dehalogenation of halothane.

Evaluation of activity of cytochrome P-450-dependent monooxygenase system induced by β-naphthoflavone and dexamethasone in rats at different ages

This study was undertaken to investigate the effects of age and some inducers (BNF-40 mg/kg/day, DEX − 20 mg/kg/day for 3 days each) on hepatic mixed function oxidase. Wistar male rats of very young (0.5 and 1 months), older (2, 4, 8,12 months) and very old (20 and 28 months) ages were used. The cytochrome P-450 content and the activity of NADH-cytochrome b5 reductase were highest in one-month-old animals, while the cytochrome b5 content was highest in the youngest but NADPH-cytochrome P-450 activity in the oldest rats. β-Naphthoflavone (BNF) treatment resulted in an enhanced content of cytochrome P-450 in all age groups but the activity NADPH-cytochrome P-450 reductase was increased only in the oldest rats. Dexamethasone (DEX) treatment led to a decrease in the activity of NADPH-cytochrome P-450 reductase in the oldest animals only. The cytochrome P-450 content was decreased in young animals (0.5-, 1-, 2-month-old), too. BNF and DEX decreased the cytochrome b 5 content and the activity of NADH-cytochrome b5 in all age groups.

Different influences of two fractions of lung cytochrome b5 on reconstituted lung benzphetamine N-demethylase system

Chromatography of lung microsomal cytochrome b5 obtained from DEAE-cellulose columns, yielded two distinct cytochrome b5 fractions. These cytochrome b5 fractions were further purified by Sephadex G-100 gel filtration chromatography. The specific cytochrome b5 content of fraction 1 and fraction 2 was found to be 16.5 and 16.4 nmol/mg protein respectively. Both fractions were free of cytochrome P-450, NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities. The effects of lung cytochrome b5 (fraction 1 and fraction 2) and liver cytochrome b5 on benzphetamine N-demethylase activity were examined. Four different reconstitution systems were used. Lung cytochrome b5 fraction 2 and liver cytochrome b5 stimulated N-demethylase activity in all four systems when b5: P-450 molar ratio was low, i.e. 0.25 or 0.5. Both cytochrome b5 samples inhibited N-demethylase activity when b5: P-450 ratio exceeded 1:1 molar ratio. In contrast lung cytochrome b5 fraction 1 stimulated N-demethylase activity in all four systems. Maximal enhancement of the activity was obtained when b5: P-450 ratio was 0.5. The greatest increase in N-demethylation activity was observed in the reconstitution system with the lowest concentration of cytochrome P-450 reductase, conditions which most closely resemble intact microsomes.

Characterization of a human bladder cancer cell line selected for resistance to BMY 25067, a novel analogue of mitomycin C.

This study describes characteristics of a human bladder cancer cell line, SCaBER/R, selected for resistance to a mitomycin C (MMC) analogue BMY 25067. The SCaBER/R cell line was isolated by repeated 24 h exposures of the parental cells to 0.09 microM BMY 25067 (IC90, 24 h drug exposure) over a period of about 180 days. Approximately 2.2-fold higher concentration of BMY 25067 was required to kill 50% of the SCaBER/R cell line compared with parental cells (p < 0.001). The IC20 and IC90 values for BMY 25067 were also significantly higher in the SCaBER/R cell line than in SCaBER. Unlike most MMC resistant cell lines, the SCaBER/R cell line displayed a marked cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and lacked cross-resistance to cisplatin, doxorubicin or VP-16. The SCaBER/R cell line also displayed a marked cross-resistance to the parent drug (MMC) and BMY 25282, another analogue of MMC. NADPH cytochrome P450 reductase activity, an enzyme implicated in bio-reductive activation of MMC, did not differ significantly in these cells. DT-diaphorase activity, another MMC activation enzyme, was significantly lower in the SCaBER/R cell line when compared to the SCaBER cells. These results suggest that relatively lower sensitivity of SCaBER/R cell line to MMC and BMY 25067 may result from impaired drug activation. Cellular levels of glutathione (GSH) and GSH-transferase (GST), which have been suggested to affect the cytotoxicity of MMC, were comparable in SCaBER and SCaBER/R cell lines. BMY 25067 induced DNA interstrand cross-links (DNA-ISC) could not be detected in either of the cell lines even at drug concentrations which produced a significant cell kill. These findings suggest that (a) cellular resistance to BMY 25067 in the SCaBER/R cell line may be due to impaired drug activation, and (b) the nature of the cytotoxic produced by BMY 25067 may be different from that of MMC.

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: Importance to reduced hepatic content of cytochrome P450 in cholestasis

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased to 32% to 52% of control, but the activities of two male sex-specific CYPs, 2C11 and 3A2, were reduced to a significantly greater extent (P < .05). The microsomal contents of CYP proteins 2C6 and 2E1 were decreased after BDL to 61% and 63% of control, whereas 2C11 and 3A2 proteins were 21% and 45% of control. Corresponding reductions of the messenger RNA (mRNA) species for CYP 2C11 (9% of control) and 3A2 (37%) were detected, whereas there was no reduction of 2C6 mRNA. These findings are consistent with downregulation of the CYP 2C11 and 3A2 genes. Nuclear run-on studies performed 3 days after BDL showed that there was a generalized impairment of gene transcription after BDL, but a disproportionate reduction in transcription of CYPs 2C11 and 3A2. A possible explanation for downregulation of CYP 2C11 and 3A2 was provided by the observation that serum estradiol concentrations were threefold greater in BDL male rats, while serum testosterone was reduced; estradiol is known to downregulate CYPs 2C11 and 3A2. It is concluded that male sex-specific CYP enzymes are decreased to a greater extent than other microsomal proteins in BDL male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: Importance to reduced hepatic content of cytochrome P450 in cholestasis

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased to 32% to 52% of control, but the activities of two male sex-specific CYPs, 2C11 and 3A2, were reduced to a significantly greater extent ( P < .05). The microsomal contents of CYP proteins 2C6 and 2E1 were decreased after BDL to 61% and 63% of control, whereas 2C11 and 3A2 proteins were 21% and 45% of control. Corresponding reductions of the messenger RNA (mRNA) species for CYP 2C11 (9% of control) and 3A2 (37%) were detected, whereas there was no reduction of 2C6 mRNA. These findings are consistent with downregulation of the CYP 2C11 and 3A2 genes. Nuclear run-on studies performed 3 days after BDL showed that there was a generalized impairment of gene transcription after BDL, but a disproportionate reduction in transcription of CYPs 2C11 and 3A2. A possible explanation for downregulation of CYP 2C11 and 3A2 was provided by the observation that serum estradiol concentrations were threefold greater in BDL male rats, while serum testosterone was reduced; estradiol is known to downregulate CYPs 2C11 and 3A2. It is concluded that male sex-specific CYP enzymes are decreased to a greater extent than other microsomal proteins in BDL male rats. It is proposed that the reduced activity of steroid metabolizing enzymes, which occurs after BDL, results in altered serum sex steroid concentrations, which, in turn, lead to decreased transcription of CYP 2C11 and 3A2 genes.

Identification of glutathione S-transferase as a determinant of 4-hydroperoxycyclophosphamide resistance in human breast cancer cells

Aldehyde dehydrogenase (ALDH) is well known for its involvement in the resistance of tumor cells to cyclophosphamide (CPA) and its activated derivatives, such as 4-hydroperoxy-CPA (4HC). The role of other drug-metabolizing enzymes such as glutathione S-transferase (GST) in CPA resistance is, however, less certain. In the present study of a human breast cancer cell line (MCF-7) exhibiting about 6-fold resistance to 4HC (MCF/HC), cellular levels of glutathione (GSH) were increased 1.4-fold, while cytosolic GST and ALDH activities were increased 2.7- and 7.2-fold, respectively, relative to the MCF-7 parental line. No significant changes in glutathione peroxidase and NADPH cytochrome P450 reductase activity, and no increase in microsomal GST and GSTπ mRNAs were found in the resistant cells. Treatment with the ALDH substrate octanal sensitized the cells to the cytotoxic effects of 4HC to a modest extent in both MCF-7 and MCF/HC cells [dose modification factor (DMF) of 1.4 and 1.6, respectively]. Depletion of GSH by treatment with the GSH synthesis inhibitor buthionine sulfoximine (BSO) enhanced the cytotoxic effect of 4HC to a similar extent in both cell lines. By contrast, ethacrynic acid, which inhibited GST activity by > 85% in MCF-7 and MCF/HC cell extracts without depletion of GSH, sensitized the resistant but not the parental cells to 4HC cytotoxicity, indicating the importance of GST as a determinant of 4HC resistance in these cells. This conclusion is supported by the observation that in MCF/HC cells, ethacrynic acid in combination with BSO increased the DMF 3-fold higher than did BSO or EA alone, while in the parental MCF-7 cells ethacrynic acid with BSO had no significant chemosensitization effect over BSO alone. These studies establish that in addition to ALDH, GST overexpression can contribute to acquired resistance of tumor cells to 4HC and, furthermore, suggest that modulators that target the GSH/GST system could be useful in overcoming CPA resistance in the clinic.

Effect of propylthiouracil treatment on NADPH-cytochrome P450 reductase levels, oxygen consumption and hydroxyl radical formation in liver microsomes from rats fed ethanol or acetone chronically

The antithyroid drug propylthiouracil (PTU) has been shown previously to reduce hepatic oxygen utilization and to protect the liver from ethanol-induced injury. The present study examined the effect of PTU on hepatic microsomal oxygen consumption and on the activities of NADPH-cytochrome P450 reductase (CYP-reductase) and cytochrome P4502E1 (CYP2E1) in rats receiving ethanol or acetone chronically. Liver microsomes from rats treated with ethanol for 29 days displayed increases in (i) O 2 consumption (70%), (ii) hydroxyl radical (OH) production (49%) and (iii) ethanol oxidation (50%). Microsomal CYP2E1 levels were increased markedly by chronic ethanol administration, while CYP-reductase was affected marginally, but not significantly ( P = 0.06). Chronic treatment with acetone for 14 days, produced similar effects, except that OH production was not enhanced. Administration of PTU (25 mg/kg/day) to ethanol- or acetone-fed rats, for 10 and 14 days, respectively, led to a marked reduction in the levels and activity of CYP-reductase, and to a decrease in the rates of microsomal O 2 consumption, OH production and ethanol oxidation, but did not lower the levels of CYP2E1 or the metabolism of the CYP2E1 substrate N, N-nitrosodimethylamine. These data suggest that the ability of PTU to protect the liver from ethanol-induced injury may be due to a reduction in the levels of CYP-reductase, thereby minimizing the enhancement of microsomal oxygen consumption and free radical generation associated with ethanol-induced CYP2E1 activity.

Copper deficiency increases cytochrome P450-dependent 7-ethoxyresorufin-O-deethylase activity in rat small intestine.

Although impaired heme synthesis during copper deficiency may limit the production and function of hemoproteins, little is known about the effects of copper deficiency on the cytochromes P450, an important family of hemoproteins, in the small intestine. A series of experiments was conducted to examine the effects of copper deficiency on cytochrome P450 content, ethoxyresorufin-O-deethylase (EROD) activity, and NADPH-cytochrome P450 reductase activity in rat small intestine. Sixteen hours after a single oral administration of 5,6-benzoflavone (BF), an inducer of cytochromes P4501A1 and P4501A2, intestinal cytochrome P450 content was elevated as indicated by the CO-difference spectrum of the reduced cytochrome and by immunoblotting using anticytochrome P4501A1/1A2. However, cytochrome P450 content, measured by either method following BF induction, was not affected by copper deficiency. Thus, copper deficiency did not impair the availability of heme for cytochrome P450 synthesis in the small intestine. Even though copper deficiency did not affect intestinal cytochrome P450 content, EROD activity, which is a cytochrome P450-associated monooxygenase activity, was 9-fold higher in copper-deficient rats compared with controls following BF treatment. The finding that copper deficiency has no effect on intestinal cytochrome P450 content suggests that the increased EROD activity results from an effect of copper deficiency on the cytochrome P450 reductase component of the monooxygenase system. Measurement of cytochrome P450 reductase activity showed a 2-fold increase in the small intestines of copper-deficient rats compared with controls. It is possible, therefore, that increased intestinal cytochrome P450 reductase activity during copper deficiency increases EROD activity by facilitating the flow of electrons to cytochrome P450 during the redox cycle that cytochrome P450 undergoes during the O-deethylation of ethoxyresorufin.

Superoxide anion production during monoelectronic reduction of xenobiotics by preparations of rat brain cortex, microvessels, and choroid plexus

Brain microsomes may produce reactive metabolites during the reductive metabolism of some xenobiotics including drugs. These reactive species can, in turn, react with molecular oxygen to form superoxide radicals (O 2 .−). We measured the rates of superoxide production by homogenates obtained from three cerebral structures, cortex plus cerebellum, choroid plexus, and microvessels. The molecules assayed were related to quinone, nitroheterocycle, and iminium chemical families. The results we obtained showed a significant correlation between the rate of superoxide anion production and the apparent kinetic parameters (log K m/ V max) of NADPH-cytochrome P450 reductase activity for these molecules, suggesting the involvement of this enzyme in xenobiotic-induced superoxide production.

Postmortem changes in drug-metabolizing enzymes of rat liver microsome

Postmortem changes in the drug-metabolizing enzymes in rat liver microsome were studied. Parameters investigated were: microsomal protein, NADPH-cytochrome P-450 reductase activity, NADH-cytochrome b 5 reductase activity, cytochrome b 5 content, cytochrome P-450 content, aminopyrine N-demethylase activity, aniline p-hydroxylase activity, p-nitroanisole O-demethylase activity, uridine diphosphate (UDP)-glucuronyl transferase activity and glutathione S-transferase activity. Nearly all the parameters based on microsomal protein decreased during autolysis and the time-dependent decrement ratios of the parameters changed by various amounts. Cytochrome b 5 content decreased more rapidly than that of other components. By 36 h post mortem, levels of cytochrome b 5 were not detectable. By 48 h post mortem, NADPH-cytochrome P-450 reductase activity decreased to 91%, NADH-cytochrome b 5 reductase activity decreased to 94%, and cytochrome P-450 content decreased to 92% of relative activities. By 48 h post mortem, aminopyrine N-demethylase activity decreased to 87%, aniline p-hydroxylase activity decreased to 98% and p-nitroanisole O-demethylase activity decreased to 75% of relative activities. The activity of p-nitroanisole O-demethylase appeared to be more stable than that of aminopyrine N-demethylase or aniline p-hydroxylase. These results demonstrate that there are multiple forms of isozymes of the cytochrome P-450-linked monooxygenase system. Hepatic transferases showed decrease patterns different to those of monooxygenases, so UDP-glucuronyl transferase activity of ∼32% of relative activity was detected at 48 h post mortem. Thus, UDP-glucuronyl transferase activity appeared to be more stable than the cytochrome P-450-linked monooxygenases. These results show that these activities and components would be useful as markers of postmortem time. The causes of the variety of instability of these enzyme systems are discussed.

Stimulatory effects of lung cytochrome b5 on benzphetamine N-demethylation in a reconstituted system containing lung cytochrome P450 LgM2

1. 1. Cytochrome b5 was partially purified from sheep lung microsomes in the presence of detergents Emuigen 913 and cholate by three consecutive DEAE-cellulose and Sephadex G-100 gel filtration chromatographies. 2. 2. The specific content ofcytochrome b5 was 16.5 nmol/mg protein and purified cytochrome b5 fractions were free of cytochrome P450, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase activities. 3. 3. The influences of increasing concentrations of lung cytochrome b5 on benzphetamine N-demethylation reactions were examined in four different reconstitution systems containing lung cytochrome P 450 LgM2, lung cytochrome P450 reductase and lipid. In each system concentration of reductase was doubled with respect to former system. 4. 4. In all systems cytochrome b 5 stimulated benzphetamine Ndemethylase activity especially when cytochrome b5 was present at 0.5:1 molar ratio with respect to cytochrome / P450 LgM2. 5. 5. Besides, the greatest fold of increase in benzphetamine N-demethylation activity due to addition of cytochrome b5 was observed in System 1 with the lowest concentration of reductase.

Flavoenzymes inhibited by indomethacin.

The effect of indomethacin on the activity of five different flavoenzymes, three dehydrogenases and six hydrosases, was determined. Indomethacin at concentration 1.0 mM inhibited the activity, in decreasing order of sensitivity, of the following flavoenzymes: D-amino acid oxidase (pig kidney), flavin-containing monooxygenases (pig liver microsomal), cyclohexanone monooxygenase (Acinetobacter), NADPH-quinone reductase (pig liver), and glutathione reductase (yeast), but it had no effect on the activity of glucose oxidase (Aspergillus) or liver microsomal NADPH-cytochrome P-450 reductase. Indomethacin was competitive with D-alanine for the D-amino acid oxidase (Ki=30 microM) and with NADPH for all other flavoenzymes sensitive to this compound (Kis 170-500 microM). While indomethacin also inhibited two of the three NAD(P)+-dependent dehydrogenases tested, the Kis were relatively high (<1, 500 microM), and of the six different hydrolases tested only one, liver microsomal esterase, was inhibited by indomethacin (Ki=600 microM). Indomethacin also inhibited aminopyrine demethylation catalyzed by the liver microsomal P-450 monooxygenase (Ki=1,000 microM). Although the exact mechanism for the inhibition of functionally different flavoenzymes sensitive to indomethacin is not known, the inhibition is probably not due to the detergent properties of this drug.

Changes of the level of cytochrome P-450 and NADPH-cytochrome P-450 reductase in rats with hereditary degeneration of the retina

In animals with hereditary degeneration of the retina the level of cytochrome P-450 in the brain microsomal fraction is found to be higher than that in healthy animals. In rats with hereditary degeneration of the retina the activity of NADPH-cytochrome P-450 reductase is unchanged in all tissues examined except for the retina, where it is markedly higher than in healthy animals on postnatal day 90.

Localization of drug-metabolizing enzyme activities to blood-brain interfaces and circumventricular organs.

The brain, with the exception of the choroid plexuses and circumventricular organs, is partially protected from the invasion of blood-borne chemicals by the specific morphological properties of the cerebral micro-vessels, namely, the tight junctions of the blood-brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood-brain interfaces, where they form an "enzymatic barrier," is tested. By using microdissection techniques or a gradient-centrifugation isolation procedure, the activities of seven drug-metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three circumventricular organs and cerebral microvessel than in the cortex. Very high membrane-bound epoxide hydrolase and UDP-glucuronosyltransferase activities (approaching those in liver) and somewhat high 7-benzoxyresorufin-O-dealkylase and NADPH-cytochrome P-450 reductase activities were determined in the choroid plexuses. The pia-arachnoid membranes, but not the dura matter, displayed drug-metabolizing enzyme activities, notably that of epoxide hydrolase. The drug-metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.

Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream ( Sparus aurata)

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2–3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M r of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 × 10 −6 M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 7-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

This study was undertaken to elucidate the mechanism(s) of differential sensitivity of human bladder cancer cell lines J82 and SCaBER to mitomycin C (MMC) and its analogue, BMY 25067. The IC50 values for MMC and BMY 25067 in the SCaBER cell line were respectively 5- and 4-fold higher than in J82. BMY 25282 and BMY 25067 were significantly more cytotoxic, on a molar basis, than MMC in both the cell lines. NADPH cytochrome P450 reductase and DT diaphorase activities were significantly higher in the J82 cell line than in SCaBER, suggesting that relatively lower sensitivity of the SCaBER cell line to MMC and BMY 25067 may be due to deficient drug activation. This conclusion was supported by the observation that IC50 values for BMY 25282, which has lower quinone reduction potential than MMC and BMY 25067, did not differ significantly in these cell lines. A correlation between drug sensitivity, oxyradical formation and levels of antioxidative enzymes was not observed. These results suggest that the relatively lower sensitivity of SCaBER cells to MMC or BMY 25067 may be independent of differential oxyradical formation. MMC-induced DNA interstrand cross-link (ISC) formation was markedly lower in the SCaBER cell line than in J82. However, it remains to be seen if the reduced ISC frequency in the SCaBER cell line is a consequence of deficient drug activation or results from increased repair of the damaged DNA.

Aryl hydrocarbon hydroxylase activity in chemically induced and toremifene‐treated mammary tumors in rats

Aryl hydrocarbon hydroxylase (AHH) and NADPH-cytochrome P450 reductase (NCR) activity of microsomes from liver, lungs, uterus and mammary tumors in dimethylbenzanthracene-induced and toremifene-treated female Sprague-Dawley rats were studied. AHH and NCR activity in tumors and uteri was low compared with that in livers and lungs. The distribution of AHH in tumors was wide and skewed. It varied in different tumors of the same animal as widely as between different animals. The enzyme activity in tumors did not correlate with that in the liver, lungs or uterus of the same animal. Toremifene had no effect on AHH or NCR in tumor or liver, but it decreased them in lungs. Tumor AHH activity correlated with its overall growth rate and development stage. The results suggest that malignant transformation leading to the defect in growth regulation also confuses the complex regulatory system of AHH activity.

Aryl hydrocarbon hydroxylase activity in spontaneous mammary tumors in rat.

The development of spontaneous mammary tumors was observed for about 2 years in a group of 25 female Sprague-Dawley rats aged over 1 year at the beginning of the study. All younger females in our animal facility were similarly monitored. In old females, the incidence of spontaneous mammary tumors was 64%. The parity of rats did not protect them from tumorigenesis, but the proportion of malignant tumors was higher in virgin (57%) than in parous (13%) rats. Activities of cytochrome P450IA1-dependent enzyme (aryl hydrocarbon hydroxylase, AHH) and NADPH-cytochrome P450 reductase (NCR) were determined in microsome fractions isolated from livers, lungs, uteri and tumors of rats. AHH and NCR activities in tumors and uteri were low compared to those in livers or lungs. In tumors, the activity distributions were wide, even in different tumors of the same animal the AHH activities varied as widely as between different animals. The activities in benign and malignant tumors were not statistically significantly different. No correlation with liver, lung or uterine activities was found either. With ageing of the rat, the AHH activities in tumors, liver and lungs decreased. The behavior of AHH in spontaneous mammary tumors in rats seems to be similar to that found in chemically induced tumors and seems to show individual regulation, possibly altered by tumorigenesis in each individual tumor.