Toxicokinetics of Chiral PCB 136 and Its Hydroxylated Metabolites in Mice with a Liver-Specific Deletion of Cytochrome P450 Reductase.

Exposure to polychlorinated biphenyls (PCBs) has been implicated in adverse human health effects, including developmental neurotoxicity. Several neurotoxic PCBs are chiral and undergo atropisomeric enrichment in vivo due to atropselective metabolism by cytochrome P450 enzymes. Here we study how the liver-specific deletion of the cytochrome P450 reductase ( cpr) gene alters the toxicokinetics of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in mice. Male and female mice with a liver-specific deletion of cpr (KO) and congenic wild-type (WT) mice were exposed to a single oral dose of racemic PCB 136 (6.63 mg/kg). Levels and chiral signatures of PCB 136 and its hydroxylated metabolites were determined 1-48 h after PCB exposure in whole blood. Blood levels of PCB 136 were typically higher in M-WT compared to F-WT mice. At the later time points, F-KO mice had significantly higher PCB 136 levels than F-WT mice. 2,2',3',4,6,6'-Hexachlorobiphenyl-3-ol (3-150), 2,2',3,3',6,6'-hexachlorobiphenyl-4-ol (4-136), 2,2',3,3',6,6'-hexachlorobiphenyl-5-ol (5-136), and 4,5-dihydroxy-2,2',3,3',6,6'-hexachlorobiphenyl (4,5-136) were detected in blood, with 5-136 and 4-136 being major metabolites. At later time points, the sum of HO-PCB (∑HO-PCB) levels exceeded PCB 136 levels in the blood; however, higher ∑HO-PCB than PCB 136 levels were observed later in KO than WT mice. PCB 136 and its major metabolites displayed atropisomeric enrichment in a manner that depended on the time point, sex, and genotype. Toxicokinetic analysis revealed sex and genotype-dependent differences in toxicokinetic parameters for PCB 136 atropisomers and its metabolites. The results suggest that mice with a liver-specific deletion of the cpr gene can potentially be used to assess how an altered metabolism of neurotoxic PCB congeners affects neurotoxic outcomes following exposure of the offspring to PCBs via the maternal diet.

Impact of hepatic P450-mediated biotransformation on the disposition and respiratory tract toxicity of inhaled naphthalene.

We determined whether a decrease in hepatic microsomal cytochrome P450 activity would impact lung toxicity induced by inhalation exposure to naphthalene (NA), a ubiquitous environmental pollutant. The liver-Cpr-null (LCN) mouse showed decreases in microsomal metabolism of NA in liver, but not lung, compared to wild-type (WT) mouse. Plasma levels of NA and NA-glutathione conjugates (NA-GSH) were both higher in LCN than in WT mice after a 4-h nose-only NA inhalation exposure at 10ppm. Levels of NA were also higher in lung and liver of LCN, compared to WT, mice, following exposure to NA at 5 or 10ppm. Despite the large increase in circulating and lung tissue NA levels, the level of NA-GSH, a biomarker of NA bioactivation, was either not different, or only slightly higher, in lung and liver tissues of LCN mice, relative to that in WT mice. Furthermore, the extent of NA-induced acute airway injury, judging from high-resolution lung histopathology and morphometry at 20h following NA exposure, was not higher, but lower, in LCN than in WT mice. These results, while confirming the ability of extrahepatic organ to bioactivate inhaled NA and mediate NA's lung toxicity, suggest that liver P450-generated NA metabolites also have a significant, although relatively small, contribution to airway toxicity of inhaled NA. This hepatic contribution to the airway toxicity of inhaled NA may be an important risk factor for individuals with diminished bioactivation activity in the lung.

P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms.

Cytochrome P450 oxidoreductase (POR) is required for metabolic reactions of steroid and drug metabolizing cytochrome P450 proteins located in endoplasmic reticulum. Mutations in POR cause a complex set of disorders resembling combined deficiencies of multiple steroid metabolizing enzymes. The P450 oxidoreductase deficiency (PORD) was first reported in patients with symptoms of defects in steroidogenic cytochrome P450 enzymes and ambiguous genitalia, and bone malformation features resembling Antley-Bixler syndrome. POR is now classified as a separate and rare form of congenital adrenal hyperplasia (CAH), which may cause disorder of sexual development (DSD). Since the initial description of PORD in 2004, a large number of POR mutations and polymorphisms have been described. In this report we have performed computational analysis of mutations and polymorphisms in POR linked to metabolism of steroids and xenobiotics and pathology of PORD from the reported cases. The mutations in POR that were identified in patients with disruption of steroidogenesis also have severe effects on cytochrome P450 proteins involved in metabolism of drugs. Different variations in POR show a range of diverse effects on different partner proteins that are often linked to the location of the particular variants. The variations in POR that cause defective binding of co-factors always have damaging effects on all partner proteins, while the mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each individual partner. Computational analysis of available sequencing data and mutation analysis shows that Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations can be linked to unique founder mutations. Other mutations identified so far were identified as rare alleles or in single isolated reports. The common polymorphism of POR is the variant A503V which can be found in about 27% of alleles in general population but there are remarkable differences among different sub populations.

Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-pyrido[2,3-b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice.

2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H), and N(2)-(ß-1-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H, and AαC-N(2)-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration-time curve (AUC0-24h) of AαC and AαC-N(2)-Glu were 4-24-fold higher in LCN than in WT mice, but they were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0-24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg kg(-1)). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ∼ 40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.

Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency.

Cytochrome P450 oxidoreductase (POR) is a 2-flavin protein that transfers electrons from NADPH via its FAD and FMN moieties to all microsomal cytochrome P450 enzymes, including steroidogenic and drug-metabolizing P450s. Defects in the POR gene can cause POR deficiency (PORD), manifested clinically by disordered steroidogenesis, genital anomalies and skeletal malformations. We examined the POR mutant A287P, which is the most frequent cause of PORD in patients of European ancestry and partially disrupts most P450 activities in vitro. Flavin content analysis showed that A287P is deficient in FAD and FMN binding, although the mutation site is distant from the binding sites of both flavins. Externally added flavin partially restored the cytochrome c reductase activity of A287P, suggesting that flavin therapy may be useful for this frequent form of PORD. Transient kinetic dissection of the reaction of POR with NADPH and the reduction in cytochrome c by POR using stopped-flow techniques revealed defects in individual electron transfer steps mediated by A287P. A287P had impaired ability to accept electrons from NADPH, but was capable of a fast FMN → cytochrome c electron donation reaction. Thus the reduced rates of P450 activities with A287P may be due to deficient flavin and impaired electron transfer from NADPH.

Cytochrome P450 oxidoreductase deficiency: rare congenital disorder leading to skeletal malformations and steroidogenic defects.

Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is a newly characterized disorder. PORD is caused by homozygous or compound heterozygous mutations in POR encoding an electron donor for several microsomal enzymes such as CYP21A2, CYP17A1, CYP19A1, CYP51A1, and CYP26A1-C1. Molecular defects of PORD include a Japanese founder mutation p.R457H, as well as various missense, nonsense, frameshift, and splice-site mutations and exonic deletions. PORD leads to unique skeletal malformations referred to as Antley-Bixler syndrome, in addition to 46,XX and 46,XY disorders of sex development, pubertal failure, adrenal dysfunction, and maternal virilization during pregnancy. Such clinical features are ascribable to impaired activities of the POR-dependent microsomal enzymes. PORD represents one form of congenital adrenal hyperplasia, although it can occur as a congenital malformation syndrome and a disorder of sex development. Phenotypic severity of PORD is highly variable and only partly depends on the residual activity of the mutant proteins. It is possible that PORD remains undiagnosed in several patients. Detailed hormonal assessment and molecular analysis are useful for diagnosis of PORD.

Mice deficient in intestinal epithelium cytochrome P450 reductase are prone to acute toxin-induced mucosal damage.

Cytochrome P450 (P450) enzymes are a superfamily of heme-containing enzymes involved in the metabolism of various endogenous compounds, including retinoids, glucocorticoids, and eicosanoids, that are postulated to participate in the maintenance and/or development of inflammatory and immune reactions in the intestinal mucosa. To investigate the role of P450 enzymes in intestinal inflammation and immunity, we took advantage of IE-Cpr-null mice, which are deficient in intestinal epithelium of NADPH-cytochrome P450 reductase (CPR), the obligate redox partner of all microsomal P450 enzymes. We report that IE-Cpr-null mice, following an acute toxin challenge, had higher levels of pro-inflammatory chemokines and increased tissue damage compared to wild-type mice. IE-Cpr-null mice had normal Peyer's patch numbers and elicited normal secretory IgA (SIgA) responses. However, SIgA baseline levels in IE-Cpr-null mice were consistently elevated over WT littermates. While neither retinoic acid nor glucocorticoid levels in serum and intestinal homogenates were altered in IE-Cpr-null mice, basal levels of arachidonic acid metabolites (11,12-DiHETE and 14,15-DiHETE) with known anti-inflammatory property were significantly lower compared to WT controls. Overall, these findings reveal immunological and metabolic changes resulting from a genetic deficiency in CPR expression in the intestine, and support a role for microsomal P450 enzymes in mucosal homeostasis and immunity.

Deficits in neuronal cytochrome P450 activity attenuate opioid analgesia but not opioid side effects.

Morphine-like analgesics act on µ opioid receptors in the CNS to produce highly effective pain relief, but the same class of receptors also mediates non-therapeutic side effects. The analgesic properties of morphine were recently shown to require the activity of a brain neuronal cytochrome P450 epoxygenase, but the significance of this pathway for opioid side effects is unknown. Here we show that brain P450 activity is not required for three of morphine׳s major side effects (respiratory depression, constipation, and locomotor stimulation). Following systemic or intracerebroventricular administration of morphine, transgenic mice with brain neuron - specific reductions in P450 activity showed highly attenuated analgesic responses as compared with wild-type (control) mice. However, brain P450-deficient mice showed normal morphine-induced side effects (respiratory depression, locomotor stimulation, and inhibition of intestinal motility). Pretreatment of control mice with the P450 inhibitor CC12 similarly reduced the analgesia, but not these side effects of morphine. Because activation of brain µ opioid receptors produces both opioid analgesia and opioid side effects, dissociation of the mechanisms for the therapeutic and therapy-limiting effects of opioids has important consequences for the development of analgesics with reduced side effects and/or limited addiction liability.

NADPH P450 oxidoreductase: structure, function, and pathology of diseases.

Cytochrome P450 oxidoreductase (POR) is an enzyme that is essential for multiple metabolic processes, chiefly among them are reactions catalyzed by cytochrome P450 proteins for metabolism of steroid hormones, drugs and xenobiotics. Mutations in POR cause a complex set of disorders that often resemble defects in steroid metabolizing enzymes 17α-hydroxylase, 21-hydroxylase and aromatase. Since our initial reports of POR mutations in 2004, more than 200 different mutations and polymorphisms in POR gene have been identified. Several missense variations in POR have been tested for their effect on activities of multiple steroid and drug metabolizing P450 proteins. Mutations in POR may have variable effects on different P450 partner proteins depending on the location of the mutation. The POR mutations that disrupt the binding of co-factors have negative impact on all partner proteins, while mutations causing subtle structural changes may lead to altered interaction with specific partner proteins and the overall effect may be different for each partner. This review summarizes the recent discoveries related to mutations and polymorphisms in POR and discusses these mutations in the context of historical developments in the discovery and characterization of POR as an electron transfer protein. The review is focused on the structural, enzymatic and clinical implications of the mutations linked to newly identified disorders in humans, now categorized as POR deficiency.

Dietary regulation of mouse intestinal P450 expression and drug metabolism.

The study was originally designed to test the hypothesis that the compensatory increase in intestinal P450 (cytochrome P450) expression in the intestinal epithelium-specific P450 reductase (CPR) knockout (IE-Cpr-null) mice was attributable to decreased metabolism of putative P450 inducers present in the diet. Thus, we determined the impact of a dietary change from regular rodent chow to a synthetic diet devoid of phytochemicals on the expression of P450 enzymes in the small intestine (SI) and liver of wild-type (WT) and IE-Cpr-null mice. The dietary change diminished expression of CYP1A, 2B, 2C, and 3A in SI and CYP2B, 2C, and 3A in liver of both WT and IE-Cpr-null mice. However, the compensatory increase in SI P450 expression still occurred in IE-Cpr-null, compared with WT, mice, on the synthetic diet. The diet change-induced decrease in P450 expression was accompanied by decreases in microsomal midazolam-hydroxylase activity in vitro and first-pass clearance of midazolam in vivo in WT mice. Further studies showed that the dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content and that treatment of WT mice on the synthetic diet with GW4064, a farnesoid-X-receptor agonist, restored the levels of CYP3A expression in both liver and SI to those seen in mice fed with regular chow. Taken together, these results highlight the vital role of diet in maintaining adequate expression of major drug-metabolizing P450s and their associated drug-metabolizing activities in the digestive tract and suggest potential involvement of BA signaling in the regulatory mechanisms.

The role of cytochrome P450-dependent metabolism in the regulation of mouse hepatic growth hormone signaling components and target genes by 3-methylcholanthrene.

3-Methylcholanthrene (MC) is a readily metabolized aryl hydrocarbon receptor (AHR) agonist. MC disrupts expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). To determine if these effects of MC depend on hepatic microsomal P450-mediated activity, we examined biologic responses to MC treatment in liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of NADPH-cytochrome P450 oxidoreductase (POR). MC caused mild induction of Por and a hepatic inflammatory marker in wild-type mice, whereas MC caused strong induction of AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 in wild-type and LCN mice. Two mouse hepatic STAT5b target genes, Cyp2d9 and major urinary protein 2 (Mup2), were suppressed by MC in wild-type mice, and the CYP2D9 mRNA response was maintained in LCN mice. In wild-type mice only, MC decreased hepatic GH receptor (GHR) mRNA but increased GHR protein levels. There was an apparent impairment of STAT5 phosphorylation by MC in wild-type and LCN mice, but large interanimal variation prevented achievement of statistical significance. In vehicle-treated mice, basal levels of MUP2 mRNA, GHR mRNA, GHR protein, and the activation status of extracellular signal-regulated kinase 2 and Akt were influenced by hepatic Por genetic status. These results indicate that the effects of MC on hepatic GH signaling components and target genes are complex, involving aspects that are both dependent and independent of hepatic microsomal P450-mediated activity.

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

Two-step biochemical differential diagnosis of classic 21-hydroxylase deficiency and cytochrome P450 oxidoreductase deficiency in Japanese infants by GC-MS measurement of urinary pregnanetriolone/ tetrahydroxycortisone ratio and 11ß-hydroxyandrosterone.

BACKGROUND: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5ß-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11ß-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11ß-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

CONTEXT: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. OBJECTIVE: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. DESIGN: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. RESULTS: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. CONCLUSIONS: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

The interplay between tubulins and P450 cytochromes during Plasmodium berghei invasion of Anopheles gambiae midgut.

BACKGROUND: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. METHODOLOGY/PRINCIPAL FINDINGS: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B) silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. CONCLUSIONS: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to Plasmodium infection.

A rare cause of congenital adrenal hyperplasia: Antley-Bixler syndrome due to POR deficiency.

Cytochrome P450 oxidoreductase (POR) deficiency is a recently discovered new variant of congenital adrenal hyperplasia. Distinctive features of POR deficiency are the presence of disorders of sexual development in both sexes, glucocorticoid deficiency and skeletal malformations similar to those observed in the Antley-Bixler syndrome.

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency.

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency.

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Discrimination of haptens from prohaptens using the metabolically deficient Cpr(low/low) mouse.

The murine local lymph node assay (LLNA) is a validated, well accepted method for identification of chemical contact allergens. Both direct acting haptens and prohaptens (requiring metabolic activation) can be identified, but not differentiated by this assay. This study was used to assess the utility of a pan microsomal metabolic deficient mouse to distinguish between direct acting haptens and prohaptens in the LLNA. Hapten and prohapten induced cell proliferation was compared in C57BL/6J (B6) wild type (WT) versus homozygous (HO) knockout mice with a hypomorphic NADPH-Cytochrome P450 Reductase (CPR) gene (termed Cpr(low/low)) resulting in low CPR enzyme activity. Mice were dosed with known prohaptens; benzo(a)pyrene (BaP), carvone oxime (COx) and paracetamol (PCM) and haptens; oxazolone (OX), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (EtOX), and N-acetylbenzoquinoneimine (NABQI) in this study. Skin microsomes from the WT, HO and heterozygous (HT) Cpr(low/low) mice were compared and evaluated for CPR activity. Lymphocyte proliferative responses to BaP, COx and PCM were significantly abrogated by 36.4%, 45.2% and 50.8%, respectively; in Cpr(low/low) knock out (KO) mice versus WT mice; while the lymphocyte proliferative responses to the direct acting haptens OX, EtOX and NABQI were comparable. CPR activity, determined as Units/mg protein, was determined to be significantly lower in the Cpr(low/low) mice compared to the WT. Results of the present study suggest potential utility of the Cpr(low/low) mice in the LLNA to differentiate prohaptens from direct acting haptens.