S. M. Nijmeijer

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.

Phase I and phase II enzyme activities in Ringed seals (Phoca hispida): characterization of hepatic cytochrome P450 by activity patterns, inhibition studies, mRNA analyses, and western blotting

Abstract Hepatic phase I and phase II enzymes play an important role in contaminant metabolism in mammals. Knowledge of these enzymes is essential since their presence and activity determines the potential biological effects of contaminant exposure. In this study activities of hepatic phase I enzymes (cytochrome P450 (CYP)) and phase II enzymes (UDP glucuronosyl transferase (UDPGT) and glutathione S -transferase (GST)) in Ringed seals ( Phoca hispida ) were assessed. In addition, CYP enzymes were characterized using catalytic activities, selective inhibitors, mRNA analyses, as well as Western blotting. Both UDPGT and GST activities were present, indicating that these seals may form the reactive methylsulfonated PCB metabolites. The results from the CYP characterization showed ethoxyresorufin- O -deethylation (EROD) and caffeine demethylation activity, while the pentoxyresorufin- O -depenthylation activity was low. The activity towards testosterone resulted in several hydroxy-metabolites. Based on these activity studies the presence of CYP1A, CYP3A, but not CYP2B was insinuated. The inhibition of EROD and caffeine demethylation by α -naphthoflavone but not by furafylline suggested that in this seal species only one CYP1A enzyme was present. This was supported by the results from the mRNA measurements and Western blots. Only one mRNA band cross-hybridized with human CYP1A cDNA probes at the rat CYP1A1 position, while also one protein band, cross reacting with anti-rat CYP1A, was detected. The selective inhibition of the formation of the testosterone 2 β - and 6 β -hydroxy metabolites by ketoconazole supported the suggestion that the formation of these metabolites was mediated by CYP3A. The mRNA measurements and the results from the Western blots confirmed these results. The Northern blots showed cross hybridization with human CYP3A cDNA, while in the Western blots one protein band cross-reacting with anti-rat CYP3A was detected. No cross hybridization with rat CYP2B1/2 cDNA was observed. However, the Western blots showed a band cross-reacting with anti-rat CYP2B, suggesting the presence of a CYP2B-like protein. In conclusion, this study has shown that Ringed seal liver contains multiple forms of CYP as well as phase II enzymes, showing different catalytic activities, i.e. EROD, caffeine- N -demethylation, and testosterone hydroxylation at different positions. Only one CYP1A isoform seemed to be present as well as a CYP3A-like isoform. Although the catalytic activities and mRNA analyses did not indicate the presence of a CYP2B-like protein in Ringed seals, the Western blots suggested the presence of a CYP2B-like enzyme. However, its functional significance remains unclear.

Time-dependent induction of two distinct hepatic cytochrome P4501A catalytic activities at low temperatures in Arctic charr (Salvelinus alpinus) after oral exposure to benzo(a)pyrene

Due to increased industrial and other anthropogenic activities during the last decades, the arctic environment faces increasing levels of organic pollution. The presence of polycyclic aromatic hydrocarbons in the arctic environment is of major concern because of their carcinogenic potential and their effect on the health and reproductive performance of animals. There is an increasing demand for sensitive and relatively inexpensive diagnostic biomarkers, applicable for environmental monitoring in the Arctic seas to assess the human impact on the Arctic. As a first step to validate the use of enzymatic assays in fish as an indicator for environmental pollution, the hepatic cytochrome P450 induction in the anadromous Spitsbergen Arctic charr was studied at several time intervals during 4 days following oral exposure to benzo(a)pyrene. Three different enzyme activities were studied, i.e. testosterone-6β-hydroxylation, the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation. Both the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation activity (only the 1,7-dimethylxanthine metabolite was formed) showed a strong and time-dependent induction after exposure. Western immunoblotting revealed an increase in the amount of protein. In the exposed groups a clear time-dependent increase of protein reacting with anti-cod CYP1A was observed. Between the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation activity in the exposed animals there was a strong and linear correlation. There was no effect of benzo(a)pyrene exposure on the testosterone-6β-hydroxylation. The slow and long lasting induction, probably due to the low water temperatures 5 °C, can be regarded as advantageous for biomonitoring. Differences in inhibition (α-naphtaflavone, furafylline) characteristics between ethoxyresorufin-O-deethylation rate and caffeine-N3-demethylation rate could be interpreted in terms of possible presence of more than one P4501A isoform in this fish species.

Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model

In breast cancer, the interaction between estrogen-producing breast adipose fibroblasts (BAFs) and estrogen-dependent epithelial tumor cells is pivotal. Local estrogen production is catalyzed by aromatase, which is differentially regulated in disease-free and tumorigenic breast tissue. The use of aromatase inhibitors to block local estrogen production has proven effective in treatment of estrogen-dependent breast cancer. However, a major problem during breast cancer treatment is the sudden onset of menopause and many women seek for alternative medicines, such as the soy isoflavone genistein. In this study, we show that genistein can induce estrogen-dependent MCF-7 tumor cell growth and increase breast cancer-associated aromatase expression and activity in vitro. We have previously developed an in vitro breast cancer model where the positive feedback loop between primary BAFs and estrogen-dependent MCF-7 tumor cells is operational, thereby representing a more natural in vitro model for breast cancer. In this model, genistein could negate the growth inhibitory action of the aromatase inhibitor fadrozole at physiologically relevant concentrations. These data suggest that soy-based supplements might affect the efficacy of breast cancer treatment with aromatase inhibitors. Considering the high number of breast cancer patients using soy supplements to treat menopausal symptoms, the increasing risk for adverse interactions with breast cancer treatment is of major concern and should be considered with care.

Endotoxin-induced liver damage in rats is minimized by β2-adrenoceptor stimulation

AbstractObjective and Design: To investigate the effects of β2-adrenoceptor (β2-AR) stimulation on endotoxin-induced liver damage and systemic cytokine levels in rats. Subjects: Standard male Wistar rats. Treatment: A disease-model of lipolysaccharide (LPS)-induced acute systemic inflammation was used. The β2-selective AR agonist clenbuterol was administered before, during, and after LPS-challenge to investigate its effects on the acute inflammatory response and associated liver-failure. Methods: The following parameters have been measured in plasma: TNFα, IL-1β, IL-6, IL-10, AST, ALT, and Bilirubin. Liver histological examination was performed to look for changes in tissue morphology. Results: Administration of clenbuterol (p.o.) one hour before, or intravenous at the same time as LPS-challenge resulted in a marked reduction of plasma levels of TNFα, IL-1β, and IL-6. A change both in plasma-level and in time-concentration profile of the anti-inflammatory cytokine IL-10 was found. Clenbuterol minimized LPS-induced liver damage, as represented by significantly lowered concentrations of several parameters for liver-failure (AST, ALT, Bilirubin), and improved hepatic tissue morphology. Clenbuterol administration after LPS challenge failed to inhibit TNFα-release but reduced liver-damage. Simultaneous use of the β2-AR antagonist propranolol augmented LPS-induced liver failure, suggesting a role of endogenous adrenoceptor-agonists in prevention of organ-failure during systemic inflammation. Conclusions: The results indicate that a selective β2-AR agonist might be used as an additional therapeutic agent in the clinic for the treatment of (acute) systemic inflammatory disorders in order to reduce or prevent subsequent liver failure.

Selective effects of a bacterial infection (Actinobacillus pleuropneumoniae) on the hepatic clearances of caffeine, antipyrine, paracetamol, and indocyanine green in the pig

1. In order to investigate the effect of a bacterial acute phase response model on drug disposition in vivo, plasma clearances of antipyrine, caffeine, paracetamol and indocyanine green were investigated in the healthy and Actinobacillus pleuropneumoniae-infected pig. 2. Indocyanine green plasma and endogenous creatinine clearance were not changed during the infection, which indicates that hepatic blood flow and renal function were not significantly affected. 3. In the A. pleuropneumoniae-infected pig, plasma clearances of antipyrine and caffeine, both marker substrates for hepatic oxidative biotransformation, were decreased by 72 and 68% respectively. The clearance of paracetamol, a drug mainly glucuronidated in the pig, was reduced by 39%. 4. It is concluded that the most important change in drug elimination during an acute phase response induced by A. pleuropneumoniae is a suppression of oxidative hepatic biotransformation.

Differential Roles of Phase I and Phase II Enzymes in 3,4-Methylendioxymethamphetamine-Induced Cytotoxicity

Metabolism plays an important role in the toxic effects caused by 3,4-methylenedioxymethamphetamine (MDMA). Most research has focused on the involvement of CYP2D6 enzyme in MDMA bioactivation, and less is known about the contribution of other cytochrome P450 (P450) and phase II metabolism. In this study, we researched the differential roles of phase I P450 enzymes CYP1A2, CYP3A4, and CYP2D6 and phase II enzymes glutathione S-transferase (GST) and catechol-O-methyltransferase (COMT) on the toxic potential of MDMA. MDMA acts as inhibitor of its own metabolism with a relative potency of inhibition of CYP2D>CYP3A≫ CYP1A in rat liver microsomes and in human liver [immortalized human liver epithelial cells (THLE)] cells transfected with individual CYP1A2, CYP3A4, or CYP2D6. Cytotoxicity measurements [by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in THLE cells showed that the inhibition of phase I enzymes CYP1A2 by α-naphthoflavone and CYP3A4 by troleandomycin does not affect MDMA-induced cytotoxicity. MDMA metabolism by CYP2D6 significantly increased cytotoxicity, which was counteracted by CYP2D6 inhibition by quinidine. Inhibition of COMT by 2′-fluoro-3,4-dihydroxy-5-nitrobenzophenone (Ro-41-0960) and GST by buthionine sulfoximine showed that COMT is mainly involved in detoxification of CYP2D6-formed MDMA metabolites, whereas glutathione (GSH) is mainly involved in detoxification of CYP3A4-formed MDMA metabolites. Liquid chromatography/tandem mass spectrometry analyses of MDMA-metabolites in the THLE cell culture media confirmed formation of the specific MDMA metabolites and corroborated the observed cytotoxicity. Our data suggest that CYP2D6 as well as CYP3A4 play an important role in MDMA bioactivation. In addition, further studies are needed to address the differential roles of CYP3A4 and GSH/GST in MDMA bioactivation and detoxification.

Species- and sex-related differences in the plasma clearance and metabolite formation of antipyrine. A comparative study in four animal species: Cattle, goat, rat and rabbit

1. The plasma disposition of antipyrine, and its urinary metabolite pattern, were studied in both sexes of four animal species: rat, dwarf goat, rabbit and cattle. 2. No sex differences in plasma elimination of antipyrine were found in rabbit and goat; however, in rat and cattle the effect of sex was marked. As expected, male rat showed a higher plasma clearance value than female. In contrast bulls showed a significantly lower clearance value than cows. 3. Metabolite patterns varied widely from one species to another. The major urinary metabolite in rabbit and the two ruminant species was 4-hydroxy-antipyrine (OHA), whereas in rat 3-hydroxymethylantipyrine (HMA) was quantitatively the most important metabolite. 4. HMA was excreted in the 24 h urine in larger amounts by male rats than by females. Metabolism of antipyrine to HMA was also sexually different in the dwarf goat, but in this species females were more active than males. The effect of sex on the metabolite pattern in cattle was marked. 5. It is concluded that in ruminants there may be xenobiotic metabolic pathways which are under hormonal control, just as there are in rats and mice. If hormones influence drug metabolism in food-producing animals, residue levels of xenobiotics or their metabolites in food from animal origin may differ with the sex of the animal, or may be altered after treatment with anabolic hormones.

Phytoestrogens in menopausal supplements induce ER-dependent cell proliferation and overcome breast cancer treatment in an in vitro breast cancer model

Breast cancer treatment by the aromatase inhibitor Letrozole (LET) or Selective Estrogen Receptor Modulator Tamoxifen (TAM) can result in the onset of menopausal symptoms. Women often try to relieve these symptoms by taking menopausal supplements containing high levels of phytoestrogens. However, little is known about the potential interaction between these supplements and breast cancer treatment, especially aromatase inhibitors. In this study, interaction of phytoestrogens with the estrogen receptor alpha and TAM action was determined in an ER-reporter gene assay (BG1Luc4E2 cells) and human breast epithelial tumor cells (MCF-7). Potential interactions with aromatase activity and LET were determined in human adrenocorticocarcinoma H295R cells. We also used the previously described H295R/MCF-7 co-culture model to study interactions with steroidogenesis and tumor cell proliferation. In this model, genistein (GEN), 8-prenylnaringenin (8PN) and four commercially available menopausal supplements all induced ER-dependent tumor cell proliferation, which could not be prevented by physiologically relevant LET and 4OH-TAM concentrations. Differences in relative effect potencies between the H295R/MCF-7 co-culture model and ER-activation in BG1Luc4E2 cells, were due to the effects of the phytoestrogens on steroidogenesis. All tested supplements and GEN induced aromatase activity, while 8PN was a strong aromatase inhibitor. Steroidogenic profiles upon GEN and 8PN exposure indicated a strong inhibitory effect on steroidogenesis in H295R cells and H295R/MCF-7 co-cultures. Based on our in vitro data we suggest that menopausal supplement intake during breast cancer treatment should better be avoided, at least until more certainty regarding the safety of supplemental use in breast cancer patients can be provided.