Mitigating role of baicalein on lysosomal enzymes and xenobiotic metabolizing enzyme status during lung carcinogenesis of Swiss albino mice induced by benzo(a)pyrene

The lungs mainly serve as a primary site for xenobiotic metabolism and constitute an important defense mechanism against inhalation of carcinogens. Our current study aimed to evaluate the chemotherapeutic efficacy of baicalein (BE) in Swiss albino mice exposed to tobacco‐specific carcinogen benzo(a)pyrene [B(a)P] for its ability to mitigate pulmonary carcinogenesis. Here, we report that altered activities/levels of lysosomal enzymes (cathepsin‐D, cathepsin‐B, acid phosphatase, β‐D‐galactosidase, β‐D‐glucuronidase, and β‐D‐N‐acetyl glucosaminidase), phase I biotransformation enzymes (cytochrome P450, cytochrome b5, NADPH‐cytochrome P450 reductase, and NADH‐cytochrome b5 reductase), and phase II enzymes (glutathione S‐transferase, UDP‐glucuronyl transferase, and DT‐diaphorase) were observed in the B(a)P‐induced mice. Treatment with BE significantly restored back the activities/levels of lysosomal enzymes, phase I and phase II biotransformation enzymes. Moreover, assessment of lysosomal abnormalities by transmission electron microscopic examination revealed that BE treatment effectively counteract B(a)P‐induced oxidative damages. Protein expression levels studied by immunohistochemistry, immunofluorescence, and immunoblot analysis of CYP1A1 revealed that BE treatment effectively negate B(a)P‐induced upregulated expression of CYP1A1. Further analysis of scanning electron microscopic studies in lung was carried out to substantiate the anticarcinogenic effect of BE. The overall data suggest that BE treatment significantly inhibits lysosomal and microsomal dysfunction, thus revealing its potent anticarcinogenic effect.     

Cytochrome P450 1A1, 2E1 and GSTM1 Gene Polymorphisms and Susceptibility to Colorectal Cancer in the Saudi Population

Background: The Saudi population has experienced a sharp increase in colorectal and gastric cancer incidenceswithin the last few years. The relationship between gene polymorphisms of xenobiotic metabolizing enzymesand colorectal cancer (CRC) incidence has not previously investigated among the Saudi population. The aimof the present study was to investigate contributions of CYP1A1, CYP2E1, and GSTM1 gene polymorphisms.Materials and Methods: Blood samples were collected from CRC patients and healthy controls and genotypeswere determined by polymerase chain reaction restriction fragment length polymorphism and sequencing.Results and Conclusions: CYP2E1*6 was not significantly associated with CRC development (odd ratio=1.29;confidence interval 0.68-2.45). A remarkable and statistically significant association was observed amongpatients with CYP1Awt/*2A (odd ratio=3.65; 95% confidence interval 1.39-9.57). The GSTM1*0/*0 genotypewas found in 2% of CRC patients under investigation. The levels of CYP1A1, CYP2E1 and GSTM1 mRNAgene expression were found to be 4, 4.2 and 4.8 fold, respectively, by quantitative real time PCR. The results ofthe present case-control study show that the studied Saudi population resembles Caucasians with respect to theconsidered polymorphisms. Investigation of genetic risk factors and susceptibility gene polymorphisms in ourSaudi population should be helpful for better understanding of CRC etiology.     

Prediction of the metabolic clearance of benzophenone-2, and its interaction with isoeugenol and coumarin using cryopreserved human hepatocytes in primary culture

Benzophenone-2 (BP2) is widely used as a UV screen in both industrial products and cosmetic formulations, where it is frequently found associated with fragrance compounds, such as isoeugenol and coumarin. BP2 is now recognized as an endocrine disruptor, but to date, no information has been reported on its fate in humans. The intrinsic clearance (Cl_int) and metabolic interactions of BP2 were explored using cryopreserved human hepatocytes in primary cultures. In vitro kinetic experiments were performed to estimate the Michaelis–Menten parameters. The substrate depletion method demonstrated that isoeugenol was cleared more rapidly than BP2 or coumarin (Cl_int = 259, 94.7 and 0.40 μl/min/10⁶ cells respectively). This vitro model was also used to study the metabolic interactions between BP2 and isoeugenol and coumarin. Coumarin exerted no effects on either isoeugenol or BP2 metabolism, because of its independent metabolic pathway (CYP2A6). Isoeugenol appeared to be a potent competitive substrate inhibitor of BP2 metabolism, equivalent to the specific UGT1A1 substrate: estradiol. Despite the fact that inhibition of UGT by xenobiotics is not usually considered to be a major concern, the involvement of UGT1A1 in BP2 metabolism may have pharmacokinetic and pharmacological consequences, due to the its polymorphisms in humans and its pure estrogenic effect.     

Induction of carbonyl reductase 1 (CBR1) expression in human lung tissues and lung cancer cells by the cigarette smoke constituent benzo[a]pyrene

Carbonyl reductase 1 (CBR1) reduces various xenobiotic carbonyl substrates to corresponding alcohol metabolites. Here we demonstrated that benzo[a]pyrene (B[a]P), a potent pro-carcinogen and predominant polycyclic aromatic hydrocarbon (PAH) compound in cigarette smoke and air pollutants, upregulates CBR1 gene expression in vitro and in vivo, and that a proximal xenobiotic response element (XRE) motif (₋₁₂₂XRE) mediates the induction effect of B[a]P. First, we observed 46% and 50% increases in CBR1 mRNA and CBR1 protein levels, respectively, in human lung tissue samples from smokers compared to never-smokers. Second, we detected 3.0-fold (p < 0.0001) induction of CBR1 mRNA and 1.5-fold (p < 0.01) induction of CBR1 protein levels in cells of the human lung cancer cell line A549 incubated with 2.5 μM B[a]P for 24 h. Third, results from experiments with CBR1 promoter constructs indicated that a proximal XRE motif (₋₁₂₂XRE) mediates induction of reporter activity in response to B[a]P. Furthermore, we detected enhanced nuclear translocation of aryl hydrocarbon receptor (AhR) following B[a]P exposure in A549 cells. Finally, we demonstrated increased binding of specific protein complexes to ₋₁₂₂XRE in nuclear extracts from B[a]P-treated cells and the presence of the AhR/Arnt complex in the specific nuclear protein ₋₁₂₂XRE complexes.     

Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study using two strains of mice with different sensitivities to dioxin

We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2–20 μg/kg), all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 μg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects.     

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.