Reem H. Elbekai

The Effect of Liver Cirrhosis on the Regulation and Expression of Drug Metabolizing Enzymes

Cirrhosis is the end stage of many forms of liver pathologies including hepatitis. The liver is known for its vital role in the processing of xenobiotics, including drugs and toxic compounds. Cirrhosis causes changes in the architecture of the liver leading to changes in blood flow, protein binding, and drug metabolizing enzymes. Drug metabolizing enzymes are primarily decreased due to loss of liver tissue. However, not all enzyme activities are reduced and some are only altered in specific cases. There is a great deal of discrepancy between various reports on cytochrome p450 alterations in liver cirrhosis, likely due to differences in disease severity and other underlying conditions. In general, however, CYP1A and CYP3A levels and related enzyme activities are usually reduced and CYP2C, CYP2A, and CYP2B are mostly unaltered. Both alcohol dehyrogenases and aldehyde dehydrogenases are altered in liver cirrhosis, although the etiology of the disease may determine the expression of alcohol dehydrogenases. Glucuronidation is mainly preserved, but there are a number of factors that determine whether glucuronidation is affected in patients with liver cirrhosis. Low sulphation rates are usually found in patients with liver disease but a decrease in sulfatase activity compensates for the decrease in sulphation rates. In all cases, a reduction in drug metabolizing enzyme activities in liver cirrhosis contributes to decreased clearance of drugs seen in patients with liver abnormalities. The reduction in drug metabolizing enzyme activity must be taken into consideration when adjusting doses, especially in patients with severe liver disease.

Benzo[a]Pyrene, 3-Methylcholanthrene and ß-Naphthoflavone Induce Oxidative Stress in Hepatoma Hepa 1c1c7 Cells by an AHR-dependent Pathway

Polycyclic aromatic hydrocarbons have been shown to cause oxidative stress in vitro and in vivo in various animal models but the mechanisms by which these compounds produce oxidative stress are unknown. In the current study we have investigated the role of the aryl hydrocarbon receptor (AHR) in the production of reactive oxygen species (ROS) by its cognate ligands and the consequent effect on cyp1a1 activity, mRNA and protein expressions. For this purpose, Hepa 1c1c7 cells wild-type (WT) and C12 mutant cells, which are AHR-deficient, were incubated with increasing concentrations of the AHR-ligands, benzo[a]pyrene (B[a]P, 0.25-25 μM), 3-methylcholanthrene (3MC, 0.1-10 μM) and β-naphthoflavone (βNF, 1-50 μM). The studied AHR-ligands dose-dependently increased lipid peroxidation in WT but not in C12 cells. However, only B[a]P and βNF, at the highest concentrations tested, significantly increased H2O2 production in WT but not C12 cells. The increase in lipid peroxidation and H2O2 production by AHR-ligands were accompanied by a decrease in the cyp1a1 catalytic activity but not mRNA or protein expressions, which were significantly induced in a dose-dependent manner by all AHR-ligands, suggesting a post-translational mechanism is involved in the decrease of cyp1a1 activity. The AHR-ligand-mediated decrease in cyp1a1 activity was reversed by the antioxidant N-acetylcysteine. Our results show that the AHR-ligands induce oxidative stress by an AHR-dependent pathway.

Regulation of CYP1A1 by heavy metals and consequences for drug metabolism.

Cytochrome P450 1A1 (CYP1A1) is a hepatic and extrahepatic enzyme that is regulated by the aryl hydrocarbon receptor signaling pathway. With the growing human exposure to heavy metals, emerging evidence suggests that heavy metals exposure alter CYP1A1 enzyme activity. Heavy metals regulate CYP1A1 at different levels of its aryl hydrocarbon receptor signaling pathway in a metal- and species-dependent manner. The importance of CYP1A1 emerges from the fact that it has been always associated with the metabolism of pro-carcinogenic compounds to highly carcinogenic metabolites. However, recently CYP1A1 has gained status along with other cytochrome P450 enzymes in the metabolism of drugs and mediating drug–drug interactions. In addition, CYP1A1 has become a therapeutic tool for the bioactivation of prodrugs, particularly cytotoxic agents. In this review, we shed light on the effect of seven heavy metals, namely arsenic, mercury, lead, cadmium, chromium, copper and vanadium, on CYP1A1 and the consequences on drug metabolism.

Effects of renal diseases on the regulation and expression of renal and hepatic drug-metabolizing enzymes: a review

The activity of drug-metabolizing enzymes (DMEs) in extrahepatic organs is highest in the kidneys. Generally, the kidneys contain most, if not all, of the DMEs found in the liver. Surprisingly, some of these DMEs show higher activity in the kidneys than in the liver. Most of the renal DMEs are localized in the cortex of the kidneys, especially in the proximal tubules. DMEs are also found in the distal tubules and collecting ducts. Renal diseases such as acute and chronic renal failure and renal cell carcinoma alter the regulation of both hepatic and extrahepatic phase I and II DMEs. Changes in the expression of these DMEs seem to be tissue and species specific. Generally, there is significant down-regulation of most of the phase I and a few of phase II DMEs at the protein, mRNA and activity levels. Unfortunately, the mechanisms leading to the alteration in DMEs in renal diseases remain unclear, although many theories have been made. The presence of some circulating factors such as cytokines, nitric oxide, parathyroid hormones and increased intracellular calcium play a role in the regulation of DMEs in renal diseases.

Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy.

Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

Incidence of Spontaneous Non-Neoplastic Lesions in Transgenic CBYB6F1-Tg(HRAS)2Jic Mice

Since 2003, the Tg.rasH2 model has been accepted by regulatory agencies worldwide for 26-week short-term carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice. However, over the decade, the number of actual studies conducted with alternative mouse models has remained low. The primary cause for low acceptance of this model has been lack of a historical database for the incidence of spontaneous lesions. Recently, we published the historical control database on spontaneous tumors in the Tg.rasH2 mice. The purpose of this publication is to present a large database pertaining to the non-neoplastic spontaneous lesions noted in Tg.rasH2 mice from studies conducted at our facility. Lesions that are considered unique in Tg.rasH2 mice are skeletal muscle myopathy, vascular anomalies involving various organs, and mesenteric arterial thrombosis. Other notable lesions are extramedullary hematopoiesis of spleen, subacute inflammatory foci in the liver, and infiltration of histiocytes in the lungs.

Assessment of the mutagenic potential of Cr(VI) in the oral mucosa of Big Blue® transgenic F344 rats.

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water was associated with an increased incidence of oral tumors in F344 rats in a 2‐year cancer bioassay conducted by the National Toxicology Program. These tumors primarily occurred at 180 ppm Cr(VI) and appeared to originate from the gingival mucosa surrounding the upper molar teeth. To investigate whether these tumors could have resulted from a mutagenic mode of action (MOA), a transgenic mutation assay based on OECD Test Guideline 488 was conducted in Big Blue® TgF344 rats. The mutagenic oral carcinogen 4‐nitroquinoline‐1‐oxide (4‐NQO) served as a positive control. Mutant frequency was measured in the inner gingiva with adjacent palate, and outer gingiva with adjacent buccal tissue. Exposure to 10 ppm 4‐NQO in drinking water for 28 days increased mutant frequency in the cII transgene significantly, from 39.1 ± 7.5 × 10−6 to 688 ± 250 × 10−6 in the gingival/buccal region, and from 49.8 ± 17.8 × 10−6 to 1818 ± 362 × 10−6 in the gingival/palate region. Exposure to 180 ppm Cr(VI) in drinking water for 28 days did not significantly increase the mutant frequency in the gingival/buccal (44.4 ± 25.4 × 10−6) or the gingival/palate (57.8 ± 9.1 × 10−6) regions relative to controls. These data indicate that high (∼180,000 times expected human exposure), tumorigenic concentrations of Cr(VI) did not significantly increase mutations in the gingival epithelium, and suggest that Cr(VI) does not act by a mutagenic MOA in the rat oral cavity. Environ. Mol. Mutagen. 56:621–628, 2015.

Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications.

NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 microM), Cd2+ (5 microM), and Cr6+ (25 microM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway.

MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells.

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 microM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content.

A robust method for assessing chemically induced mutagenic effects in the oral cavity of transgenic Big Blue® rats.

The Big Blue® (BB) in vivo mutation assay uses transgenic rodents to measure treatment‐induced mutations in virtually any tissue. The BB assay can be conducted in rats or mice and is ideal for investigating tissue‐specific mutagenic mode of action of tumor induction. Some tissues such as oral mucosa have not been thoroughly studied. Due to the small quantity and cartilaginous nature of oral cavity tissues, development of special prosection and DNA isolation methods was required to permit robust analysis of mutations in these tissues. Improved surgical methods permitted collection of adequate and reproducible quantities of tissue (∼45 mg gingiva/buccal and ∼30 mg gingiva/palate). Optimized DNA isolation methods included use of liquid nitrogen pulverization, homogenization, nuclei pelleting, digestion, and phenol/chloroform extraction, to yield sufficient quantities of DNA from these tissues. In preliminary optimization work, mutant frequency (MF) in tongue and gingiva was increased in rats exposed to the promutagen, benzo[a]pyrene, and the direct mutagen, N‐ethyl‐N‐nitrosourea. The oral cavity carcinogen, 4‐nitroquinoline‐1‐oxide (4‐NQO; 10 ppm in drinking water; 28 days), was qualified as a positive control for mutagenesis in oral tissues since it caused significant increases in cII MFs in gingiva/palate (50.2‐fold) and gingiva/buccal tissues (21.3‐fold), but not in liver or bone marrow (0.9‐ and 1.4‐fold, respectively). These results are consistent with the observation that 4‐NQO primarily induces tumors in oral cavity. Results also demonstrate the utility of the BB rat mutation assay and optimized methods for investigation of oral cavity mutagenicity, and by extension, analysis of other small and cartilaginous tissues. Environ. Mol. Mutagen. 56:629–636, 2015.