Stelvio M. Bandiera

The monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450

This review examines the monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450 (P450)1 enzymes and their mechanisms of action in archaeal, bacterial, and mammalian systems. In the P450 catalytic cycle, a transient iron oxo monooxygenating species is generated that reacts with substrate to produce a monooxygenated product. We describe results of early investigations that endeavored to trap and detect this elusive monooxygenating species, as well as results of experiments that attempted to generate and characterize this active oxidant spectroscopically after reacting ferric P450 enzymes with peroxy compounds (e.g. peroxides, peracids) or single oxygen atom donors (e.g. periodate, iodosobenzene). Surrogate oxidants were able to promote P450-catalyzed monooxygenations in a manner similar to that of O2/NAD(P)H, suggesting involvement of a common transitory monooxygenating species in the two pathways. This common P450 oxidant was characterized as a porphyrin radical iron(IV) oxo complex and assigned a Compound I structure (Por+FeIV=O) exhibiting a formal FeV oxidation state. Other reactive oxidants, such as the ferric oxenoid complex (PorFeIII=O), ferryloxy radical species (PorFeIV-O·), and perferryloxo entity (PorFeV=O), were also proposed to function as P450 monooxygenating species. We also discuss the possible involvement of the ferriperoxo (PorFeIII-OO-) and ferrihydroperoxo (PorFeIII-OOH) species as alternative oxidants in P450-mediated monooxygenation reactions.

Comparison of polychlorinated dibenzodioxin levels with hepatic mixed‐function oxidase induction in great blue herons

As part of the Canadian Wildlife Service monitoring of great blue herons in British Columbia, eggs were collected from three colonies with low, intermediate, and high levels of PCDD and PCDF contamination: Nicomekl, Vancouver, and Crofton, respectively. One egg from each nest was used for chemical analysis by GC-MS; the others were hatched. Liver microsomes were prepared from the heron chicks and used for determination of cytochrome P-450-dependent activities. No erythromycin N-demethylase activity was found in any sample. Ethoxyresorufin O-dealkylase activity in the Nicomekl group was similar to that in pigeons, a control altricial species. The ethoxyresorufin activity in the herons from the Crofton colony was 2.6-fold higher than in the Nicomekl group. The Vancouver colony was intermediate. No difference among the three heron colonies was found in pentoxyresorufin O-dealkylase activity, although levels were 20-33 times that in the pigeon. Chemical analysis was carried out on paired heron eggs. Vancouver and Crofton eggs contained 13.5 and 21 times the levels of 2,3,7,8-TCDD compared to the Nicomekl group. The Crofton eggs contained higher levels of several other contaminants also. A highly significant correlation (p less than .001) was found between ethoxyresorufin O-dealkylase and 2,3,7,8-TCDD concentrations. The correlation coefficient did not change when ethoxyresorufin O-dealkylase was compared to total chemical contamination using several toxic equivalency factors. Multiple regression analysis resulted in only one predictor variable for ethoxyresorufin O-dealkylase: 2,3,7,8-TCDD.

Dioxin contamination and growth and development in great blue heron embryos

A great blue heron colony located near a pulp mill in British Columbia failed to fledge young in 1987, with a concurrent sharp increase in polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels in their eggs. In 1988 we tested the hypothesis that the PCDD and PCDF contamination caused reproductive failure by increasing mortality of the heron embryos in ovo. Pairs of great blue heron eggs were collected from three British Columbia colonies with low, intermediate, and high levels of dioxin contamination: Nicomekl, Vancouver, and Crofton, respectively. One egg of each pair was incubated under laboratory conditions at the University of British Columbia (UBC) while the other egg was analyzed for PCDDs and PCDFs. All incubated eggs were fertile. All eggs from the Nicomekl colony hatched, while 13 of 14 eggs from Vancouver and 12 of 13 eggs from Crofton hatched. Subcutaneous edema was observed in 4 of 12 chicks from Crofton and 2 of 13 chicks from Vancouver. No edema was seen in the chicks from Nicomekl. There was a small, but significant, negative regression of plasma calcium concentration, yolk-free body weight, tibia length, wet, dry, and ash weight, beak length, and kidney and stomach weight of the hatched chicks on the tetrachlorodibenzo-p-dioxin (TCDD) level of the paired eggs. Fewer down follicles were present on the heads of TCDD-contaminated chicks. Hence while dioxins did not cause mortality of the heron embryos in ovo, the depression of growth and the presence of edema are suggestive that dioxins at the levels found in the environment have an adverse effect on the development of great blue heron embryos.

Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat

The effects of exogenous hormone treatment on the expression of cytochromes P450 2C7 and P450 2C11 were studied in neonatally gonadectomized and sham-operated male and female rats. Hepatic levels of cytochrome P450 2C7 were found to be two- to threefold higher in intact adult female versus male rats. Neonatal gonadectomy resulted in a reversal of the relative cytochrome P450 2C7 levels in male and female animals at maturity. Expression of this isozyme was restored in ovariectomized females by estradiol treatment. Furthermore, neonatal and/or pubertal administration of estradiol to intact male rats induced cytochrome P450 2C7 to adult female levels. On the other hand, administration of testosterone at all times examined had no effect in intact female rats, but decreased cytochrome P450 2C7 to normal levels in neonatally castrated males treated during adulthood. Neonatal testosterone treatment also increased hepatic cytochrome P450 2C7 content in both ovariectomized females and intact males. These results indicate that estrogen is required for full expression of cytochrome P450 2C7 while the effect of testosterone is ambiguous. In comparison, neonatal gonadectomy of male rats abolished the adult expression of cytochrome P450 2C11. Normal levels were restored only by treatment with testosterone during adulthood. Neonatal testosterone treatment did not induce cytochrome P450 2C11 levels in gonadectomized rats of either sex. In contrast, neonatal estrogen treatment suppressed cytochrome P450 2C11 expression in intact adult male rats to the same extent as neonatal castration. These results indicate that androgen exposure during the adult, and not the neonatal, phase is essential for full expression of cytochrome P450 2C11.

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.

Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour

To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.

Catalytic and immunologic characterization of hepatic and lung cytochromes P450 in the polar bear

The Arctic Ocean is subject to considerable influx of anthropogenic pollutants including halogenated organic compounds. The polar bear (Ursus maritimus) is at the top of the arctic marine food web and is an ideal species for monitoring the level and distribution of contaminants in the arctic ecosystem. As the first step in the development of a biological method for assessing the functional exposure of polar bears to xenobiotics, biochemical studies were undertaken to characterize polar bear cytochromes P450. Liver and lung samples were obtained in the field from four, freshly killed, adult, male polar bears and immediately frozen at -196 degrees. Microsomes were subsequently prepared and used for the measurement of total cytochrome P450 content and aminopyrine N-demethylase, benzphetamine N-demethylase, ethylmorphine N-demethylase, p-nitrophenol hydroxylase and testosterone hydroxylase activities. Immunoblots containing hepatic and lung microsomal samples from the polar bears were probed using antibodies generated against several purified rat cytochrome P450 isozymes. Monoclonal antibody to rat cytochrome P450 1A1 and polyclonal antibodies to rat cytochromes P450 1A1, 2B1 and 3A1, as well as antibody to epoxide hydrolase, cross-reacted to varying degrees with polar bear hepatic microsomes. In addition, polyspecific antibody to the rat cytochrome P450 2C subfamily gave several immunostained protein bands, but antibodies specific to rat cytochrome P450 2C7 and 2C13 did not react, while antibody specific to cytochrome P450 2C11 yielded an ambiguous result. Except for anticytochrome P450 2B1 and polyspecific antibody to the cytochrome P450 2C subfamily, the antibodies listed above did not cross-react with polar bear lung microsomes at the protein concentrations used. The results demonstrate that polar bear liver contains multiple forms of cytochrome P450 that are catalytically active toward diverse substrates and that several of these forms are immunochemically related to rat cytochrome P450 isozymes. Immunochemical homologues of rat cytochrome P450 1A, 2B, 2C and 3A subfamilies, and of rat epoxide hydrolase are present in polar bear liver. In addition, the polar bears all had high levels of immunoreactive cytochrome P450 1A and 2B proteins, probably as a consequence of induction by environmental contaminants.

Monooxygenase, Peroxidase and Peroxygenase Properties and Reaction Mechanisms of Cytochrome P450 Enzymes

This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

Developmental Expression and Endocrine Regulation of CYP1B1 in Rat Testis

Mammalian testis expresses xenobiotic-metabolizing enzymes, including cytochrome P450 1B1 (CYP1B1), which catalyzes the bioactivation of procarcinogens and other chemicals. The factors that control testicular expression of CYP1B1 are largely not known. In the present study, we investigated the influence of age and pituitary, gonadal, and thyroid hormones on CYP1B1 expression in rat testis. Immunoblot analysis showed that testicular CYP1B1 protein was expressed at a level of 5.9 ± 2.0 (mean ± S.E.M.) pmol/mg microsomal protein in prepubertal 22-day-old rats, whereas it was 6.6-fold greater in pubertal rats (34 days old) and 9.6-fold greater in adult rats (84–91 days old). Hypophysectomy decreased testicular CYP1B1 protein levels by 69% in adult rats when compared with intact rats of the same age. Intermittent subcutaneous administration of growth hormone to hypophysectomized adult rats further decreased it by 63%. Luteinizing hormone (LH) and follicle-stimulating hormone increased CYP1B1 expression in hypophysectomized rats, but they did not restore protein levels to those in intact adult male rats. Prolactin treatment alone had no effect; however, it potentiated the increase in CYP1B1 mRNA and protein expression by LH. 3,5,3′-Triiodothyronine, but not thyroxine, resulted in a small increase in testicular CYP1B1 protein levels. Likewise, treatment of hypophysectomized rats with testosterone propionate elicited a small increase in CYP1B1 protein expression. In contrast, treatment of intact adult male rats with 17β-estradiol benzoate decreased it by 91%. Overall, our findings indicate that rat testicular CYP1B1 protein expression is subject to developmental and endocrine control, with multiple hormones playing a role.

Expression of HSP70 and CYP1A protein in ovary and liver of juvenile rainbow trout exposed to β-naphthoflavone

Cytochrome P4501A (CYP1A) and the 70-kDa stress protein (HSP70) were determined using Western blotting in the ovary and liver of juvenile female rainbow trout (Oncorhynchus mykiss) exposed for 4 days to beta-naphthoflavone (betaNF) following a single intraperitoneal injection. Ovarian CYP1A protein was observed in both control and betaNF-exposed fish, indicating constitutive and inducible expression of CYP1A in immature trout ovaries. CYP1A protein levels determined using densitometry were 14- and 46-fold greater in betaNF-exposed trout compared to controls in the liver and ovary, respectively. Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity, a specific catalytic marker of CYP1A, was also induced 38-fold above controls following betaNF exposure. Hepatic HSP70 protein expression was significantly higher in whole cell homogenates, but not in cytosolic fractions, collected from betaNF-exposed fish in comparison to control fish. There was no difference in ovarian HSP70 levels determined in whole cell homogenates between control and betaNF-exposed fish. The observation that unlike liver, ovarian HSP70 expression remained unchanged following induction of CYP1A protein may be related to the sensitivity of the teleost ovary to environmental toxicants that act as aryl hydrocarbon receptor agonists.