John G. Lamb

Mushroom Ganoderma lucidum Prevents Colitis-Associated Carcinogenesis in Mice

Background Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice. Methods/Principal Findings Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6-phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly down-regulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue. Conclusions Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer.

Nanosilver Particle Effects on Drug Metabolism In Vitro

Nanosilver particles are present in consumer and health care products. Their effects on human microsomal cytochrome P450 (P450) activities and induction in luciferase reporter-engineered Caco-2 (MDR1.C) and HepG2 (DPX2 and 1A2DRE) cells have been investigated. The LD50 values were ∼4 μg silver/ml for HepG2 and 5 μg/ml for Caco-2 cells. At silver concentrations that showed no decreased cell viability (<1 μg silver/ml), the pregnane X receptor (PXR)-driven 4.5-fold induction response of MDR1.C cells to 50 μM omeprazole was unaffected. In DPX2 cells, the PXR-driven 5.5- and 6.5-fold induction responses to omeprazole and 10 μM rifampicin were attenuated to 4- and 3.5-fold, respectively. Nanosilver particles alone showed no induction. In 1A2DRE cells, the aryl hydrocarbon receptor-driven 5.5-fold induction response to omeprazole was attenuated to 4-fold. In 1A2DRE cells, nanosilver alone elicited slight induction at 1 μg/ml. The inhibition of human P450-selective activities by nanosilver particles in vitro was proportional to the silver/microsomal protein ratio. At a fixed (0.5 mg/ml) protein concentration, P450-selective activities differed in sensitivity (IC50 value). Coumarin 7-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation exhibited the highest IC50 values (33.5 and 31.9 μM, respectively) and S-mephenytoin 4-hydroxylation exhibited the lowest (6.4 μM). Other IC50 values were, in ascending order, 8.0 to 9.3 μM (testosterone 6β-hydroxylation, 7-benzyloxyquinoline debenzylation, and diclofenac 4-hydroxylation), 16.0 μM (chlorzoxazone 6-hydroxylation), 21.2 μM [7-methoxy-4-(aminomethyl)-coumarin O-demethylation], and 24.4 μM (7-methoxyresorufin O-demethylation). An investigation of 70 μM nanosilver particles showed that microsomal NADPH cytochrome c reductase activities were inhibited <12%. From our in vitro observations, we extrapolated that nanosilver particles reaching the liver may be a potential source of drug-drug interactions.

Drug metabolizing enzyme induction by benzoquinolines, acridine, and quinacrine; Tricyclic aromatic molecules containing a single heterocyclic nitrogen

Rats were treated with nitrogen-containing phenanthrene (3,4-, 5,6-, or 7,8-benzoquinoline) or anthracene (acridine or quinacrine) derivatives at a dose of 75 mg/kg, daily for 3 days. The hepatic drug metabolizing enzyme response ranged from no induction (quinacrine) through low (5,6-benzoquinoline), intermediate (acridine), and high (3,4-benzoquinoline) magnitude increases of only phase II enzymes, to induction of both phase I and phase II enzymes (7,8-benzoquinoline). The phase I enzyme response of 7,8-benzoquinoline was an induction of CYP1A. All three benzoquinolines, but neither anthracene derivative, elevated NAD(P)H quinone oxidoreductase activity. A similar pattern but of lesser magnitude was seen with glutathione S-transferase activity. 3,4-Benzoquinoline was the only agent to significantly increase microsomal epoxide hydrolase activity (2,3-fold). Both 3,4- and 7,8-benzoquinoline increased UDP-glucuronosyltransferase activity toward 4-nitrophenol (40% and 70%, respectively), but only the 3,4-isomer increased activity toward morphine (75%), diclofenac (75%), and testosterone (23%), and only the 7,8-isomer increased activity toward chloramphenicol (105%). 3,4-Benzoquinoline elevated the hepatic mRNA concentration of UGT2B1 but not UGT1*6. Acridine treatment increased UDP-glucuronosyltransferase activity toward morphine (47%), 1-naphthol (28%), testosterone (19%), and estrone (19%). Quinacrine failed to elevate any UDP-glucuronosyltransferase activity and depressed activities toward testosterone and estrone by 20%. This study shows that some tricyclic aromatic compounds containing a single heterocyclic nitrogen atom have the potential for use as chemoprotective agents based upon their ability to selectively induce only phase II enzymes.

Xenobiotic Metabolism of Plant Secondary Compounds in Oak (Quercus Agrifolia) by Specialist and Generalist Woodrat Herbivores, Genus Neotoma

The challenge of consuming plant compounds that are recognized to have toxic physiological effects is an unavoidable consequence of an herbivorous diet and requires mechanisms to metabolize and eliminate them after consumption. We took a pharmacological approach to understanding how an oak (Quercus agrifolia) specialist (Neotoma macrotis) and generalist (N. lepida) herbivores process the same dietary toxins. Oak contains polyphenolic compounds considered toxic to most other mammals. N. macrotis includes up to 85% of oak in their diet. N. lepida includes oak as a portion of the diet but is considered a generalist in areas where sympatric with N. macrotis. Xenobiotic metabolizing enzyme activities of N. macrotis and N. lepida were investigated after animals were fed a 70% oak diet and a toxin-free control diet. Biotransformation activities of five major enzymes [cytochrome P450s (CYP), NAD(P)H/quinone oxidoreductase (QOR), UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and glutathione S-transferase (GST)] and three specific CYP isozymes (CYP1A, CYP2B, and CYP3A) were investigated. The results indicate that, with the exception of CYP2B induction, N. macrotis and N. lepida enzyme activities are not changed by an oak diet. The major differences in enzyme activities were constitutive. The specialist, N. macrotis, had higher constitutive activity of QOR, UGT, and GST. The generalist, N. lepida, had higher constitutive activity levels of CYP1A and SULT.

3-Methylindole is Mutagenic and a Possible Pulmonary Carcinogen

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5 microM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25 microM caused apoptosis, which became extensive at 100 microM, similar to the response seen with 10 microM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.

Comparative analysis of human CYP3A4 and rat CYP3A1 induction and relevant gene expression by bisphenol A and diethylstilbestrol: Implications for toxicity testing paradigms

Bisphenol A (BPA) and diethylstilbestrol (DES) are endocrine-disrupting chemicals that interact with the human pregnane X receptor (PXR). CYP3A4 enzyme is essential in the hydroxylation of steroid hormones and is regulated by PXR. In the present study, human and rat hepatoma cell lines were exposed to BPA and DES. Both BPA and DES (10-50μM) caused a significant activation of the CYP3A4 promoter via the PXR in the DPX2 human hepatoma cell line. No activation of rat PXR was seen. BPA and DES treated DPX2 cells demonstrated increased expression of CYP3A4 mRNA, and increased enzyme activity. In summary, BPA, in concentrations relevant to current safety levels of human exposure, activates the human PXR and demonstrates an increase in CYP3A4 mRNA expression and enzyme activity. BPA actions in this model system occur to a greater extent than DES. This study raises concerns regarding our current toxicity testing paradigms and species utilization.

“Pharm‐Ecology” of Diet Shifting: Biotransformation of Plant Secondary Compounds in Creosote (Larrea tridentata) by a Woodrat Herbivore, Neotoma lepida

Diet switching in mammalian herbivores may necessitate a change in the biotransformation enzymes used to process plant secondary compounds (PSCs). We investigated differences in the biotransformation system in the mammalian herbivore, Neotoma lepida, after a radical shift in diet and secondary compound composition. Populations of N. lepida in the Mojave Desert have evolved over the past 10,000 years to feed on creosote (Larrea tridentata) from an ancestral state of consuming juniper (Juniperus osteosperma). This dietary shift represents a marked change in the dietary composition of PSCs in that creosote leaves are coated with phenolic resin, whereas juniper is high in terpenes but lacks phenolic resin. We quantified the enzyme activity of five major groups of biotransformation enzymes (cytochrome P450s, NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation, and glucuronidation) recognized for their importance to mammalian biotransformation for the elimination of foreign compounds. Enzyme activities were compared between populations of Mojave and Great Basin woodrats fed control and creosote diets. In response to creosote, the Mojave population had greater levels of cytochrome P450s (CYP2B, CYP1A) and glutathione conjugation liver enzymes compared with the Great Basin population. Our results suggest that elevated levels of cytochrome P450s and glutathione conjugation enzymes in the Mojave population may be the underlying biotransformation mechanisms that facilitate feeding on creosote.

Preclinical evaluation of 2,2,3,3-tetramethylcyclopropanecarbonyl-urea, a novel, second generation to valproic acid, antiepileptic drug

2,2,3,3-Tetramethylcyclopropanecarbonylurea (TMCU) is an amide derivative of a tetramethylcyclopropyl analogue of valproic acid (VPA), one of the leading antiepileptic drugs. Structural considerations used in the design of TMCU aimed to enhance the anticonvulsant potency of VPA and to prevent its two life-threatening side effects; i.e., teratogenicity and hepatotoxicity. The anticonvulsant activity of TMCU was evaluated in the MES, scMet, 6-Hz, scBic and scPic tests, and also in the hippocampal kindling model of partial seizures and lamotrigine-resistant amygdala kindling model of therapy-resistant seizures. Minimal motor impairment was determined using the rotorod test in mice and the positional sense test, muscle tone test, and gait and stance test in rats. The antinociceptive effect of TMCU was evaluated in the mouse formalin model of acute-tonic pain. The molecular mechanisms of action of TMCU were investigated in electrophysiological studies using the whole-cell patch-clamp technique. Teratogenicity studies were performed in a SWV/Fnn-mouse model of VPA-induced teratogenicity. TMCU hepatotoxicity was evaluated following 1-week intraperitoneal and oral administration of 50, 250 and 500 mg/kg doses to rats. In the hepatotoxicity study the blood levels of TMCU were evaluated at day 1 and day 7 of the treatment. TMCU mutagenicity was evaluated in the Ames test.

Comparison of detoxification enzyme mRNAs in woodrats (Neotoma lepida) and laboratory rats.

To understand how mammalian herbivores process plant secondary compounds, we examined differences in expression of biotransformation enzyme mRNAs among populations of wild woodrats (Neotoma lepida) and laboratory rats. We compared expression of mRNAs for 10 biotransforming enzymes in five families (CYP, mEH, QOR, GST, and UGT) by using Northern blot analysis. We found significant differences in eight of 10 mRNAs tested. We suggest that the differences in mRNA expression among populations of woodrats and laboratory rats may be due to differences in the secondary compound composition of their diets. Our results provide background for future studies of detoxification strategies in mammalian herbivores that combine pharmacological techniques with an ecological perspective.

Potent Mutagenicity of 3-Methylindole Requires Pulmonary Cytochrome P450-Mediated Bioactivation: A Comparison to the Prototype Cigarette Smoke Mutagens B(a)P and NNK

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.