The exposure to dioxin-like (DL) compounds, an important class of persistent environmental pollutants, results in the altered expression of target genes. This occurs through the binding to the aryl hydrocarbon receptor (AhR), the subsequent dimerization with the AhR nuclear translocator (ARNT), and the binding of the complex to DNA responsive elements. A number of genes are up-regulated, including, among others, the AhR repressor (AHRR) and several biotransformation enzymes, such as the members of CYP1 family and NAD(P)H-quinone oxidoreductase (NOQ1). The expression and the inducibility of the above genes were investigated in mitogen-stimulated cultured blood lymphocytes from cattle, which represent a notable source of DL-compound human exposure through dairy products and meat. As assessed by real-time PCR, all the examined genes except CYP1A2 and NQO1 were detected under basal conditions. Cell exposure to the DL-compounds PCB126 or PCB77 in the 10(-6)-10(-9)M concentration range resulted in a 2-4-fold induction of CYPIA1 and CYP1B1, which was antagonized by α-naphthoflavone or PCB153. This study demonstrates for the first time the presence and inducibility of the AhR pathway in easily accessible cells like bovine peripheral lymphocytes and prompts further investigations to verify whether similar changes could occur under in vivo conditions.

Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYPB1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity.

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole anthelmintics) known to be metabolized by those enzymes.

The cytochromes P450 (CYP) constitute a multigene family of enzymes playing a critical role in the oxidation of many endogenous and xenobiotic substrates. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and aryl amines. Three members of the CYP1 family, CYP1A1, CYP1A2, and CYP1B1, have been identified in mammals. We report here on the identification and cloning of cytochrome P4501B-like sequences from two teleost fish species and a marine mammal. Sequences clustering with CYP1B1 in phylogenetic analysis were obtained from liver cDNA of scup (Stenotomus chrysops), genomic DNA of plaice (Pleuronectes platessa), and liver cDNA of striped dolphin (Stenella coeruleoalba).

CYP1B1 is a dioxin-inducible enzyme belonging to the cytochrome P450 superfamily. It has been observed to be important in a variety of developmental processes including in utero development of ocular structures. Owing to its role in the developmental biology of eye, its dysfunction can lead to ocular developmental defects. This has been found to be true and CYP1B1 mutations have been observed in a majority of primary congenital glaucoma (PCG) patients from all over the globe. Primary congenital glaucoma is an irreversibly blinding childhood disorder (onset at birth or early infancy) typified by anomalous development of trabecular meshwork (TM). How CYP1B1 causes PCG is not known; however, some basic investigations have been reported. Understanding the CYP1B1 mediated etiopathomechanism of PCG is very important to identify targets for therapy and preventive management. In this perspective, we will make an effort to reconstruct the pathomechanism of PCG in the light of already reported information about the disease and the CYP1B1 gene. How to cite this article: Faiq MA, Dada R, Qadri R, Dada T. CYP1 B1-mediated Pathobiology of Primary Congenital Glaucoma. J Curr Glaucoma Pract 2015;9(3):77-80.

The essence of pharmacogenetic studies is to provide a scientific explanation for personal differences in the process of drugs metabolism. Polymorphism of drugs biotransformation is clinically significant from the point of view of the effectiveness and safety of pharmacotherapy and an increased risk of some illnesses. The paper presents molecular mechanisms and clinical implication of several genetic polymorphisms of cytochrome P-450 (CYP) xenobiotics metabolizing enzymes and genetic predisposition to the occurrence of some diseases.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).

In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5'flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E. coli beta-galactosidase lacZ gene. When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c. The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1+ cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol. It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element. This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.

Rats, with and without overnight fasting, were anaesthetised for 5, 15 and 30 min with diethyl ether, killed immediately and total glutathione (total GS), thiobarbituric acid-reacting substances (TBAR), radical-trapping activity (RTA), total cytochrome P450 (CYP), and 7-ethoxyresorufin O-deethylase (CYP1), 7-pentoxyresorufin O-dealkylase (CYP2B) and 4-nitrophenol hydroxylase (CYP2E1) activities of liver and kidney determined. Liver, after ether anaesthesia, but no fasting, showed 30-60% losses of total GS, RTA, and total CYP, after 5, 15 and 30 min of anaesthesia, while TBAR increased 10-, 20- and 35-fold for the same periods. Liver after ether anaesthesia and overnight fasting showed 50-85% losses of total GS, RTA and total CYP, for 0, 5, 15 and 30 min of anaesthesia, while TBAR increased 4-, 30-, 40- and 60-fold for the same periods of anaesthesia. Kidney changes were similar to those in liver. Liver CYP1 and CYP2B were decreased by 45% and 35%, respectively for 30 min of anaesthesia in fed rats, and by 80% and 30% respectively for 30 min of anaesthesia in fasted rats; in contrast, liver CYP2E1 was increased 30% by fasting alone and 70% by fasting plus 5 min of ether anaesthesia. Kidney CYP1 and CYP2B were similarly decreased by ether anaesthesia (70% and 50% respectively) in both fed and fasted rats, and CYP2E1 was similarly increased (by 40-90% in fed and 30-110% in fasted rats). The decrease in tissue total GS, RTA, total CYP, CYP1 and CYP2B, and the increase in lipid peroxidation products (TBAR), are all considered to be due to generation of reactive oxygen species and oxidative stress, associated with the increase in CYP2E1 activity that results from both fasting and exposure to diethyl ether.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants that produce cognitive and behavioral changes in children exposed during gestation and lactation. Coplanar PCBs bind the aryl hydrocarbon receptor (AHR) and can be sequestered in liver by cytochrome P450 1A2 (CYP1A2). The AHR is a ligand-activated transcription factor which increases expression of the CYP1 family, including CYP1A2. Our previous work examining genetic susceptibility to developmental PCB neurotoxicity showed that Ahr(b)Cyp1a2(-/-) mice with the high-affinity Ahr(b) allele and lacking CYP1A2 were most susceptible while Ahr(b)Cyp1a2(+/+) and poor-affinity Ahr(d)Cyp1a2(+/+) mice were resistant. To follow up, a fourth line of mice was generated with the Ahr(d)Cyp1a2(-/-) genotype and compared with the background strain Ahr(b)Cyp1a2(+/+). Dams received a PCB mixture or the corn oil vehicle at gestational Day 10 (GD10) and postnatal Day 5 (PND5). Offspring were tested at PND60 in open field locomotor, acoustic startle with pre-pulse inhibition (PPI), novel object recognition and Morris water maze. Locomotor activity was increased in PCB-treated Ahr(b)Cyp1a2(+/+) mice, but no differences were seen in control vs. PCB-treated Ahr(d)Cyp1a2(-/-) mice. PCB-treated Ahr(d)Cyp1a2(-/-) mice had a higher baseline startle response and significantly reduced pre-pulse inhibition at the 74dB level compared with corn oil-treated controls (P<0.05). PCB-treated Ahr(d)Cyp1a2(-/-) mice had impairments in novel objective recognition (P<0.05) and during all three hidden platform phases of Morris water maze (P<0.01). Combined with our previous findings, these results indicate Cyp1a2 genotype is more important in susceptibility to PCB-induced deficits in learning and memory, but Ahr genotype appears more important when assessing acoustic startle-PPI and locomotor activity.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Ascorbic acid (AsA), chlorophyll and carotenoid contents and their associated gene expression patterns were analysed in Actinidia chinensis 'Hongyang' outer pericarp. The results showed chlorophyll degradation during fruit development and softening, exposed the yellow carotenoid pigments. LHCB1 and CLS1 gene expressions were decreased, while PPH2 and PPH3 gene expressions were increased, indicating that downregulation of chlorophyll biosynthesis and upregulation of its degradation, caused chlorophyll degradation. A decrease in the expression of the late carotenoid biosynthesis and maintenance genes (LCYB1, LCYE1, CYP1, CYP2, ZEP1, VDE1, VDE2, and NCED2) and degradation gene (CCD1), showed biosynthesis and degradation of carotenoid could be regulatory factors involved in fruit development. Most genes expression data of L-galactose and recycling pathway were agreement with the AsA concentrations in the fruit, suggesting these are the predominant pathways of AsA biosynthesis. GMP1, GME1 and GGP1 were identified as the key genes controlling AsA biosynthesis in 'Hongyang' outer pericarp.

Benzo[a]pyrene (B[a]P) and related procarcinogens found in cigarette smoke and roasted foodstuff require metabolic activation to build mutagenic DNA adducts that may cause tumor diseases like colorectal cancer. The major B[a]P-activating enzymes belong to the cytochrome-P450 (CYP)-1 family and are regulated by the aryl hydrocarbon receptor (AhR). Previous studies have indicated that an inhibition of AhR is accompanied with a reduced metabolic activation of B[a]P and therefore may act protective against carcinogenesis. We investigated if the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG), a known AhR inhibitor, is able to influence B[a]P-metabolizing and B[a]P-transporting enzymes in human Caco-2 colon carcinoma cells. Strikingly, treatment with EGCG did neither affect constitutive and B[a]P-inducible expression of CYP1A1 and UDP-glucuronosyltransferase (UGT)-1A1 nor overall CYP1 and UGT enzyme activities, indicating that EGCG does not antagonize the AhR in Caco-2 cells. Since flavonoids were also identified to enhance the activity of B[a]P-carrying transporter, we analyzed if EGCG exposure alters cellular excretion of B[a]P conjugates. In contrast to the positive control fisetin, EGCG did not affect cellular excretion of B[a]P metabolites. Our data provide evidence that EGCG does not alter the metabolism and transport of B[a]P in Caco-2 cells, and thus may not protect against procarcinogenic food contaminants.

BACKGROUND

Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation.

METHODS

The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation.

RESULTS

The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of β-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, β-naphthoflavone (βNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by βNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2.

CONCLUSIONS

The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Background. The results of previous studies on the association between polymorphisms of CYP1A1 and CYP1B1 and prostate cancer (PCa) susceptibility are inconsistent. The aim of the present study was to conduct a meta-analysis in order to better estimate this association. Methods. A systematic search was carried out on PubMed, Embase, Cochrane Library, and China National Knowledge Infrastructure (CNKI) databases for relevant articles published up to 15 August 2018. Pooled odds ratios (ORs) and 95% confidence intervals were obtained using fixed-effect or random-effect models. Results. A significant association was found between the CYP1A1 rs1048943 polymorphism and PCa in the overall population (B [the minor allele] vs. A [the major allele]: OR = 1.20, 95% confidence interval (CI) = 1.04-1.39, P=0.014; AB vs. AA: OR = 1.24, 95% CI = 1.02-1.51, P=0.029; BB + AB vs. AA: OR = 1.25, 95% CI = 1.04-1.50, P=0.018) and Asian population (B vs. A: OR = 1.32, 95% CI = 1.11-1.56, P=0.001; BB vs. AA: OR = 1.81, 95% CI = 1.20-2.72, P=0.005; AB vs. AA: OR = 1.30, 95% CI = 1.03-1.64, P=0.029; BB + AB vs. AA: OR = 1.38, 95% CI = 1.11-1.73, P=0.004; BB vs. AA + AB: OR = 1.58, 95% CI = 1.08-2.01, P=0.019), but not in the Caucasian population. Moreover, we found that the rs4646903 polymorphism was associated with a significant increase in the risk of PCa in the Asian population (AB vs. AA: OR = 1.43, 95% CI = 1.13-1.80, P=0.003) and Caucasian population (BB vs. AA: OR = 2.12, 95% CI = 1.29-3.49, P=0.003). Conclusion. This meta-analysis revealed a clear association between rs1048943 and rs4646903 polymorphisms of the CYP1A1 gene but not between CYP1B1 rs10012, rs162549, rs1800440, and rs2551188 polymorphisms and the risk of PCa.

Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.

A number of xenobiotic-inducible cytochrome P450s (CYPs) are now known to be localized in the mitochondrial compartment, though their pharmacological or toxicological roles remain unclear. Here, we show that BNF treatment markedly inhibits liver mitochondrial O2 consumption rate (OCR), ADP-dependent OCR, and also reserve OCR, in wild-type mice but not in Cyp1a1/1a2(-/-) double knockout mice. BNF treatment markedly affected mitochondrial complex I and complex IV activities and also attenuated mitochondrial gene expression. Furthermore, under in vitro conditions, BNF treatment induced cellular ROS production, which was inhibited by mitochondria-targeted antioxidant Mito-CP and CYP inhibitor proadefin, suggesting that most of the ROS production was intramitochondrial and probably involved the catalytic activity of mitochondrial CYP1 enzymes. Interestingly, our results also show that the AHR antagonist resveratrol, markedly attenuated BNF-induced liver mitochondrial defects in wild-type mice, confirming the role of AHR and AHR-regulated CYP1 genes in eliciting mitochondrial dysfunction. These results are consistent with reduced BNF-induced mitochondrial toxicity in Cyp1a1/1a2(-/-) mice and elevated ROS production in COS cells stably expressing CYP1A1. We propose that increased mitochondrial ROS production and respiratory dysfunction are part of xenobiotic toxicity. Resveratrol, a chemopreventive agent, renders protection against BNF-induced toxicity.

The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ∼65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 α-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism.

Imperatorin (IM) and isoimperatorin (ISOIM) are major active components of common herbal medicines from Umbelliferae plants, and widely used in clinic. This article studies the inhibitory effect of IM and ISOIM on the activity of cytochrome P450 (CYP) enzyme, and assesses their potential drug-drug interaction. IM and ISOIM were incubated separately with human or rat liver microsomes for 30 min, with phenacetin, bupropion, tolbutamide, S-mephenytoin, dextromethorphan and midazolam as probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS, and IC50 values were calculated to assess the inhibitory effect of the two drugs on human CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 enzymes, as well as on rat CYP1A2, 2B6, 2D2 and 3A1/2, and grade their inhibitory intensity. In human liver microsomes, IM and ISOIM showed different inhibitory effects on all of the six CYP isoenzymes. They were strong inhibitors for 1A2 and 2B6. The IC50 values were 0.05 and 0.20 micromol x L(-1) for 1A2, and 0.18 and 1.07 micromol x L(-1) for 2B6, respectively. They also showed moderate inhibitory effect on 2C19, and weak effect on 2C9, 2D6 and 3A4. In rat liver microsomes, IM and ISOIM were identified as moderate inhibitors for 1A2, with IC50 values of 1.95 and 2.98 micromol x L(-1). They were moderate and weak inhibitors for 2B6, with IC50 values of 6.22 and 21.71 micromol x L(-1), respectively. They also had weaker inhibitory effect on 2D2 and 3A1/2. The results indicated that IM and ISOIM had extensive inhibitory effects on human CYP enzymes. They are strong inhibitors of CYP1 A2 and 2B6 enzymes. However, it is worth noting the interaction arising from the inhibitory effect of CYP enzymes in clinic.

Although it is well established that human cytochrome P450 1 (CYP1) family enzymes are induced by cigarette smoking through activation of the Ah receptor (AhR), it is not known whether this leads to increased metabolic activation or detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAH), which are present in cigarette smoke and the general environment. We gave oral doses of deuterated phenanthrene ([D10]Phe), a non-carcinogenic surrogate of carcinogenic PAH such as benzo[a]pyrene, to smokers (N=170, 1 or 10 μg doses) and non-smokers (N=57, 1 μg dose). Bioactivation products (dihydrodiol and tetraol) and detoxification products (phenols) of [D10]Phe were determined in 6-hour urine to obtain a comprehensive metabolic profile. Cigarette smoking increased the bioactivation of [D10]Phe, and decreased its detoxification resulting in significantly different metabolic patterns between smokers and non-smokers (p<0.01), consistent with increased cancer risk in smokers. The phenanthrene bioactivation ratios ( [D10]PheT/total [D9]OHPhe) were significantly higher (2.3 (p<0.01) to 4.8 (p<0.001) fold) in smokers than non-smokers. With solid human in vivo evidence, our results for the first time demonstrate that cigarette smoking enhances the metabolic activation of phenanthrene, structurally representative of carcinogenic PAH, in humans, strongly supporting their causal role in cancers caused by smoking. The results suggest potential new methods for identifying smokers who could be at particularly high risk for cancer.

Keywords: CYP1 enzymes; cigarette smoking; deuterated phenanthrene; metabolic activation; polycyclic aromatic hydrocarbons.

We have previously reported the synthesis and breast cancer selectivity of (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (ANI-7) in cancer cell lines. To further evaluate the selectivity of ANI-7, we have expanded upon the initial cell line panel to now include the breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, MDA-MB-231); normal breast cells (MCF-10A); and cell lines derived from colon (HT29), ovarian (A2780), lung (H460), skin (A431), neuronal (BE2C), glial (U87, SJG2), and pancreatic (MIA) cancers. We now show that ANI-7 is up to 263-fold more potent at inhibiting the growth of breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468) than normal breast cells (MCF-10A) or cell lines derived from other tumor types. Measures of growth inhibition, cell cycle analysis, morphologic assessment, Western blotting, receptor binding, gene expression, small interfering RNA technology, reporter activity, and enzyme inhibition assays were exploited to define the mechanism of action of ANI-7. In this work, we report that ANI-7 mediates its effects via the activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent induction of CYP1-metabolizing mono-oxygenases. The metabolic conversion of ANI-7 induces DNA damage, checkpoint activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer cell lines. Basal expression of AhR, the AhR nuclear translocator, and the CYP1 family members do not predict for sensitivity; however, inherent expression of the phase II-metabolizing enzyme sulfur transferase 1A1 does. For the first time, we identify (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile as a new AhR ligand.

A novel endophytic actinomycete, designated strain LD22^T, was isolated from moss [Physcomitrium sphaericum (Ludw) Fuernr] collected from Yunnan Province, Southwest China. A polyphasic taxonomic study was carried out to establish the status of this strain. Morphological and chemotaxonomic characteristics of strain LD22^T confirmed the affiliation of the isolate to the genus Actinomadura. The diamino acid present in the cell wall is meso-diaminopimelic acid. Glucose, madurose, galactose and ribose occur in whole cell hydrolysates. The menaquinones were identified as MK-9(H₄), MK-9(H₈), MK-9(H₆) and MK-9(H₂). The polar lipid profile was found to contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The major fatty acids were found to be C_16:0, 10-methyl C_18:0 and C_18:1 ω9c. The DNA G + C content of the draft genome sequence, consisting of 10.0 Mbp, was 72.5%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain LD22^T belongs to the genus Actinomadura with the highest sequence similarity to Actinomadura montaniterrae CYP1-1B^T (99.2%). However, phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate formed a phyletic line with Actinomadura rudentiformis HMC1^T (98.6%). The low level of DNA-DNA relatedness and some different phenotypic characteristics allowed the strain to be distinguished from the above-mentioned two strains. Therefore, it is concluded that strain that strain LD22^T represents a novel species of the genus of Actinomadura, for which the name Actinomadura physcomitrii sp. nov. is proposed. The type strain is LD22^T (= CCTCC AA 2018050^T = JCM 33455^T).

Cytochrome P450 isozymes (CYPs) from the CYP1 and CYP2 families located primarily in extra-hepatic tissues represent ideal candidates for chemotherapeutic drug development because: 1.) They are usually involved in the metabolism of endogenous substrates that are important for cell homeostasis and growth 2.) The over-expression of certain CYPs has been reported in various malignancies 3.) There has been much clinical success with inhibitors of CYPs involved in hormone synthesis. The most ideal candidates for chemotherapeutic drug development will be discussed in terms of their biological importance and relevant substrates. This review will focus on: 1.) CYP1A1 and CYP1B1 from the CYP1 family because of the dual role these enzymes play in the bioactivation of known carcinogens and endogenous compounds. 2.) The targeting of CYPs in hypoxic environments as a therapeutic strategy. 3.) CYP2J2 and its role in the metabolism of arachidonic acid to epoxyeicosatrienoic acids and angiogenesis will also be examined. While much progress has been made towards understanding the role of CYPs in extrahepatic tissue, future studies focused on the development of selective inhibitors coupled with appropriate delivery systems that would target the tumor micro-environments could lead to significant advancement in chemotherapeutic strategies.

CYP1B1 is a member of the CYP1 subfamily of CYP superfamily of enzymes, which contains three members, CYP1A1, CYP1A2, and CYP1B1. CYP1B1 is expressed in both adult and fetal human extrahepatic tissues, including the parenchymal and stromal cells of most organs. Mutations in the CYP1B1 gene are linked to the development of primary congenital glaucoma in humans. However, the underlying mechanisms remain unknown. Using Cyp1b1-deficient mice, we showed that CYP1B1 is constitutively expressed in retinal vascular cells with a significant role in retinal neovascularization during oxygen-induced ischemic retinopathy. We also showed CYP1B1 is constitutively expressed in trabecular meshwork (TM) cells and its expression plays a significant role in the normal development and function of the TM tissue. We have observed that germline deletion of Cyp1b1 is associated with increased oxidative stress in the retinal vascular and TM cells in culture, and retinal and TM tissue in vivo. We showed increased oxidative stress was responsible for altered production of the extracellular matrix proteins and had a significant impact on cellular integrity and function of these tissues. Collectively, our studies have established an important role for CYP1B1 expression in modulation of tissue integrity and function through the regulation of cellular redox homeostasis and extracellular microenvironment.