Ecotoxicity of the lipid-lowering drug bezafibrate on the bioenergetics and lipid metabolism of the diatom Phaeodactylum tricornutum.

Pharmaceutical residues impose a new and emerging threat to the marine environment and its biota. In most countries, ecotoxicity tests are not required for all pharmaceutical residues classes and, even when mandatory, these tests are not performed using marine primary producers such as diatoms. These microalgae are among the most abundant class of primary producers in the marine realm and key players in the marine trophic web. Blood-lipid-lowering agents such as bezafibrate and its derivatives are among the most prescribed drugs and most frequently found human pharmaceuticals in aquatic environments. The present study aims to investigate the bezafibrate ecotoxicity and its effects on primary productivity and lipid metabolism, at environmentally relevant concentrations, using the model diatom Phaeodactylum tricornutum. Under controlled conditions, diatom cultures were exposed to bezafibrate at 0, 3, 6, 30 and 60 µg L-1, representing concentrations that can be found in the vicinity of discharges of wastewater treatment plants. High bezafibrate concentrations increased cell density and are suggested to promote a shift from autotrophic to mixotrophic metabolism, with diatoms using light energy generated redox potential to breakdown bezafibrate as carbon source. This was supported by an evident increase in cell density coupled with an impairment of the thylakoid electron transport and consequent photosynthetic activity reduction. In agreement, the concentrations of plastidial marker fatty acids showed negative correlations and Canonical Analysis of Principal coordinates of the relative abundances of fatty acid and photochemical data allowed the separation of controls and cells exposed to bezafibrate with high classification efficiency, namely for photochemical traits, suggesting their validity as suitable biomarkers of bezafibrate exposure. Further evaluations of the occurrence of a metabolic shift in diatoms due to exposure to bezafibrate is paramount, as ultimately it may reduce O2 generation and CO2 fixation in aquatic ecosystems with ensuing consequences for neighboring heterotrophic organisms.

Determinação de fármacos em mananciais do estado de São Paulo e estudo da sua ecotoxicidade sobre a cianobactéria Microcystis aeruginosa / Pharmaceuticals determination in São Paulo stare springs and evaluation of their toxicity in cyanobacterium Microcystis aeruginosa

A contaminação de corpos d'água por fármacos é um tema de extrema relevância, tendo em vista problemas como a escassez de água, florações de cianobactérias tóxicas e lançamentos clandestinos de efluentes domésticos. Sendo assim, este trabalho teve como objetivo determinar a presença de cafeína (CAF), fluoxetina (FLX), levotiroxina (LVX) e bezafibrato (BZF) em mananciais do estado de São Paulo, bem como avaliar a toxicidade desses compostos à cianobactéria Microcystis aeruginosa LTPNA 08. Um método por LC-MS/MS foi desenvolvido e validado, de acordo com a RDC nº 166 da ANVISA, para a detecção de CAF, FLX, LVX e BZF em amostras ambientais. As represas Guarapiranga e Billings, bem como os rios Taiçupeba, Sorocaba, Baixo Cotia, Grande e Paraíba foram monitorados de abril a setembro de 2017. A toxicidade dos fármacos foi avaliada por meio do monitoramento do crescimento, produção de microcistinas e viabilidade celular da cianobactéria M. aeruginosa LTPNA 08. CAF foi detectada em todas as amostras analisadas, com concentrações que variaram de 6,6 ng.L-1 a 16,47 µg.L-1. No Rio Cotia foram verificadas as maiores concentrações de CAF, FLX e BZF (16,47 µg.L-1; 3,5 ng.L-1 e 322 ng.L-1, respectivamente). A LVX, cujos produtos de biotransformação não foram monitorados, não foi detectada em nenhuma amostra analisada. A concentração de 50 µg.L-1 de FLX inibiu o crescimento da cianobactéria em 82,3% (CE50: 31,4 µg.L-1). Em relação à produção de microcistinas totais, os fármacos inibiram a liberação da fração extracelular para a maior concentração testada ao longo do tempo de monitoramento, embora não tenham demonstrado efeito sobre a viabilidade celular. Sendo assim, considerando-se que fármacos estão presentes nos mananciais monitorados no estado de São Paulo e que a FLX pode causar efeito sobre a M. aeruginosa, os efeitos decorrentes da exposição a concentrações ambientais contínuas e cumulativas de fármacos em corpos d'água devem ser estudados. Além disso, uma vez que a ocorrência destas substâncias e outros contaminantes antropogênicos no ambiente aquático natural é uma questão emergente devido aos efeitos adversos potenciais que estes compostos representam para a vida aquática e os seres humanos, os tipos e níveis destes compostos, que têm um impacto maior na qualidade da água, deve ser constantemente monitorada. Práticas de gestão que investem em saneamento e na redução da descarga de efluentes não tratados, e um plano de proteção de recursos hídricos com o objetivo de garantir a segurança da água seriam medidas essenciais para reduzir o aporte de contaminantes nos corpos d'água do estado de São Paulo

Contamination of water bodies by drugs is a subject of extreme relevance considering related problems such as water scarcity, harmful cyanobacterial blooms and discharge of untreated domestic effluents. Therefore, the aim of this work was to determine the presence of caffeine (CAF), fluoxetine (FLX), levothyroxine (LVX) and bezafibrate (BZF) in springs in the State of São Paulo, and to evaluate the toxicity of these compounds in cyanobacteria Microcystis aeruginosa LTPNA 08. A LC-MS/MS method was developed and validated according to RDC nº 166 of ANVISA to assess the concentration of CAF, FLX, LVX and BZF in environmental samples. Guarapiranga and Billings reservoirs, as well as the Taiçupeba, Sorocaba, Baixo Cotia, Grande and Paraíba rivers were monitored from April to September 2017.The drugs toxicity in M. aeruginosa LTPNA 08 was assessed by monitoring their effects on cyanobacterial growth, microcystins production and cell viabilityby flow cytometry. CAF was detected in all analyzed samples at concentrations ranging from 6.6 ng to 16.47 µg.L-1.Among studied sites, Cotia river showed the highest concentrations of CAF, FLX and BZF (16.47 µg.L-1, 3.5 ng.L-1 and 322 ng.L-1, respectively). LVX, which biotransformation products were not monitored, was not detected in any of the analyzed samples. Regarding the drugs toxicity, 50 µg.L-1 of FLX inhibited the cyanobacterial grow thin 82.3% (EC50 of 31.4 µg.L-1). Although no effect on cell viability was seen by flow cytometry, the highest concentrations of all compounds tested were able to inhibit the release of microcystins. Therefore, considering that some of the drugs monitored showed to be present in water sources in São Paulo State and that FLX affects cyanobacteria M. aeruginosa growth, the effects of continuous and cumulative exposure at environmental drug concentrations of in water bodies should be evaluated. Also, since the occurrence of these substances and other anthropogenic contaminants in the natural aquatic environment is an emerging issue due to the potential adverse effects these compounds pose to aquatic life and humans, thet ypes and levels of these compounds, which have a greater impact on water quality, should be constantly monitored. Management practices investing in sanitation and in reducing discharge of untreated effluents, as well as a plan for water resources protection with the goal of ensuring water security would be essential measures in reducing drugs loading into water bodies situated in São Paulo State

Uptake and effects of a mixture of widely used therapeutic drugs in Eruca sativa L. and Zea mays L. plants.

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

Bezafibrate causes depression of the immune response and increases the sensitivity to endotoxin in association with low level of HDL and PPARα activity in hypertensive ISIAH rats.

We studied the effect of bezafibrate on hepatic PPARα activity and immune parameters in hypertensive ISIAH rats in comparison with normotensive WAG rats under conditions of LPS treatment. Bezafibrate increased activity of PPARα in WAG rats, but not in ISIAH rats. As differentiated from WAG rats, bezafibrate produced a potent effect on the content of T cell subpopulations in the thymus and spleen of ISIAH rats. Administration of LPS after injection of bezafibrate caused death of 50% ISIAH animals (but not WAG rats), which was associated with low level of HDL cholesterol and increased triglyceride content. Our results suggest that the hypolipidemic treatment (e.g., bezafibrate) can increase the severity of complications in patients with infectious and inflammatory diseases in association with low level of HDL.

Toxicity of mixtures of perfluorooctane sulphonic acid with chlorinated chemicals and lipid regulators.

The toxicological interaction of perfluorooctane sulphonic acid (PFOS) with the chlorinated pollutants triclosan and 2,4,6-trichlorophenol and the lipid regulators gemfibrozil and bezafibrate was evaluated using the combination index-isobologram equation. The endpoint for bioassays was the growth rate inhibition of the green alga Pseudokirchneriella subcapitata. The results showed that most of the binary combinations assayed exhibited antagonism at all effect levels. The addition of a third component induced a less antagonistic or even synergistic behaviour. This was particularly marked for the ternary mixture of triclosan and 2,4,6-trichlorophenol with PFOS, for which synergism was very strong at all effect levels, with a combination index as low as 0.034 ± 0.002 at EC(50) for the mixture. The results obtained indicate that the evaluation of mixture toxicity from single component data using the concentration addition approach could severely underestimate combined toxicity.

Exposure to human pharmaceuticals Carbamazepine, Ibuprofen and Bezafibrate causes molecular effects in Dreissena polymorpha.

Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels.

Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).

Fibrates are pharmaceuticals commonly used to control hypercholesterolemia in humans and they are frequently detected in the freshwater environment. Since cholesterol is the precursor of all steroid hormones, it is suspected that low cholesterol levels will impact steroidogenesis. However, the effect of fibrates on fish reproductive endocrinology is not clear; therefore the aim of the present study was to evaluate the effect of bezafibrate (BZF) on gonadal steroidogenesis and spermatogenesis of zebrafish (Danio rerio). For this purpose, adult males were exposed orally to 1.7, 33 and 70 mg BZF/g food for 21 days. Blood and gonads were collected after 48 h, 7 days and 21 days to evaluate plasma cholesterol and plasma 11-ketotestosterone (11-KT). The expression of gonadal genes involved in the steroidogenesis was quantified to determine a potential mechanism of action, likewise the effect on spermatogenesis was evaluated by examining gonadal histopathology. A time dependent monotonic decrease in the plasma cholesterol concentration was observed in fish exposed to BZF. Plasma 11-KT decreased significantly after 21 days of exposure in fish exposed to the high concentration of BZF. Different gene expression patterns were observed: down-regulation in ppara and pparg mRNA levels was observed in fish exposed to the higher concentrations after 48 h; however, the expression of pparg increased after 21 days. After 21 days an increase in the star and cyp17a1 mRNA expression was observed in fish exposed to 70 mg BZF/g food. Sampling time and bezafibrate concentration explained 52.4% and 20%, respectively, of the gene expression variability. Gonadal histology revealed the presence of germ cell syncytia in the tubular lumen of fish exposed to bezafibrate and also an increased number of cysts containing spermatocytes, which indicate testicular degeneration. The study shows that bezafibrate exerts a hypocholesterolemic effect in adult male zebrafish and its potential as an endocrine disruptor due to its effect on the gonadal steroidogenesis and spermatogenesis.

Evaluation of zebrafish DNA integrity after exposure to pharmacological agents present in aquatic environments.

Over the past few years, the increasing and uncontrolled use of pharmaceutical substances in agriculture, fish farming, human health and in veterinary medicine, together with an improper use of out-of-date medicines, has led to a consequent increase in the environmental problems linked to their disposal. In some Italian waste water treatment plants were found furosemide, a diuretic; ranitidine, an antiulcer drug; bezafibrate, a lipid regulator and ibuprofen, a painkiller. The present paper shows, by means of the synergic application of three tests (the Comet Test, the Diffusion Assay and the RAPD-PCR technique), how the DNA of zebrafish can be damaged after exposure to the above mentioned drugs. The data from the Comet Test, the Diffusion Assay and the RAPD-PCR technique were generally in agreement; these results show that all four drugs are genotoxic.

Bezafibrate induces myotoxicity in human rhabdomyosarcoma cells via peroxisome proliferator-activated receptor alpha signaling.

Fibrates, the ligands of peroxisome proliferator-activated receptor alpha (PPARalpha), are used as a class of lipid-lowering drugs in clinical practice for the treatment of dyslipidemia. Fibrates are well tolerated in most cases concomitantly with occasional adverse reactions including muscular toxicity, which is enhanced by the combination with statins. This study was designed to investigate the effects of bezafibrate as a PPARalpha agonist on human embryo rhabdomyosarcoma (RD) cells and possible mechanisms responsible for bezafibrate-mediated myopathy. The results revealed that bezafibrate caused a dose-dependent decrease in cell viability, which was fortified in association with atorvastatin at a pharmacological dose. Bezafibrate at toxic doses of 300 and 1000microM upregulated PPARalpha at the mRNA level, counteracted by a PPARalpha antagonist (MK886). Bezafibrate at a toxic dose induced typical apoptotic characteristics related to the inhibition of phosphorylation of Akt which was blocked by PPARalpha antagonist. Toxic doses of bezafibrate initiated a significant increase in pyruvate dehydrogenase kinase 4 mRNA and protein levels, compromised by MK886. These results suggest the critical roles of PPARalpha signaling in bezafibrate-induced myotoxicity and the involvement of apoptosis through Akt pathway.

Effects of lipid-lowering pharmaceuticals bezafibrate and clofibric acid on lipid metabolism in fathead minnow (Pimephales promelas).

The lipid-lowering agents bezafibrate and clofibric acid, which occur at concentrations up to 3.1 and 1.6 microg/L, respectively, are among the most frequently found human pharmaceuticals in the aquatic environment. In contrast to knowledge about their environmental occurrence, little is known about their effects in the environment. The aim of the present study was to analyze effects of these lipid-lowering agents in fish by focusing on their modes of action, lipid metabolism. Fathead minnows were exposed in aquaria to measured concentrations of 0.1, 1.27, 10.18, 101.56, and 106.7 mg/L bezafibrate and to 1.07, 10.75, and 108.91 mg/L clofibric acid for 14 and 21 d, respectively. After exposure, fish liver was analyzed for expression of peroxisome proliferator-activated receptor alpha (PPARalpha) by quantitative polymerase chain reaction (PCR), and the PPAR-regulated enzyme fatty acyl-coenzyme-A oxidase (FAO) involved in fatty acid oxidation. Bezafibrate had no effect, either on PPARalpha expression or on FAO activity, at all concentrations. In contrast, clofibric acid induced FAO activity in male fathead minnows at 108.91 mg/L. No increase in expression of PPARalpha messenger ribonucleic acid was observed. Egg production was apparently decreased after 21 d of exposure to 108.91 mg/L clofibric acid. The present study demonstrates that bezafibrate has very little or no effect on PPARalpha expression and FAO activity, but clofibric acid affects FAO activity.

Effects and interactions in an environmentally relevant mixture of pharmaceuticals.

With the goal of assessing the environmental risk of pharmaceuticals, we have previously observed that a mixture of 13 different drugs at environmentally relevant concentrations had adverse consequences on human and zebra fish cells in vitro. Here we aimed to identify both main and interaction effects within the same environmentally relevant mixture of pharmaceuticals. We studied in vitro cytotoxicity in Escherichia coli, human embryonic HEK293, and estrogen-responsive OVCAR3 tumor cells using fractional-factorial experimental design. Our approach identified a subset of compounds of primary environmental concern, namely atenolol, bezafibrate, ciprofloxacin, and lincomycin, that had statistically significant effects on prokaryotic and eukaryotic cells at environmentally relevant exposure levels (ng/l). Drugs could interact and behave as chemosensitizers, with joint effects representing a statistically significant element of mixture toxicity. Effects and interactions were concentration dependent, confirming the difficulty of dose extrapolation in mixture toxicity data. This study suggests that a thorough investigation of mixture effects can direct environmental concerns toward a handful of pharmaceuticals, which may represent an actual risk at environmental concentrations. We indicate that risk identification may strongly depend on the use of environmentally relevant exposure scenarios. Antagonistic-synergistic interactions and dose dependency of effects may hamper the modeling and prediction of mixture toxicity with pharmaceuticals. Hazard identification for micropollutants depends heavily on appropriate study designs, and we indicate the use of in vitro cytotoxicity threshold and statistical design of experiments (DOEs) as a valid approach.

Effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, Mytilus galloprovincialis.

Fibrates are hypolipidemic pharmaceuticals that have been detected as contaminants in wastewaters and surface waters. In this work, the possible effects of two fibrates, Bezafibrate (BEZA) and Gemfibrozil (GEM) in the bivalve mollusc Mytilus spp were investigated. In the immune cells, the hemocytes, addition of both compounds in vitro induced rapid lysosomal membrane destabilization, extracellular lysozyme release, NO production and decreased phagocytic activity. The effect of fibrates were partly mediated by activation of ERK and p38 MAPKs (Mitogen Activated Protein Kinases), as demonstrated by the use of specific inhibitors of different kinases. The effects of fibrates on hemocyte function were confirmed in vivo, in the hemocytes of mussels injected with 0.01, 0.1 and 1 nmol/animal (corresponding to nominal concentrations of 3.61, 36.18 and 361.8ng/g dry weight for BEZA and of 2.50, 25.03 and 250.35 ng/g dry weight for GEM, respectively) and sampled at 24h post-injection. Both compounds induced a concentration-dependent lysosomal destabilization and extracellular lysozyme release; an increase in phagocytosis was observed at the highest concentration. In vivo exposure to fibrates also induced significant effects on mussel digestive gland, the key metabolic organ in bivalves. Both BEZA and GEM increased the activity of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK), and of Glutathione transferase (GST) glutathione reductase (GSR), and total glutathione content. A significant increase in the peroxisomal enzyme catalase was observed; however, BEZA exposure decreased Palmytoyl CoA oxidase activity, whereas GEM was ineffective. The results indicate that in mussels environmental concentrations of hypolipidemic drugs can affect the immune function, as well as glycolysis, redox balance and peroxisomal function.

Bezafibrate removal by means of ozonation: primary intermediates, kinetics, and toxicity assessment.

Bezafibrate (BZF) is a lipid regulator largely used for the treatment of hyperlipidaemia. As a result of its wide use, unmetabolized BZF is released in the environment with potential toxic effects for aquatic living organisms. The results obtained in this work show that ozonation is an efficient method to degrade BZF: after 10 min of treatment (corresponding to a dose of 0.73 mmol L(-1) of ozone), the complete BZF abatement is achieved, starting from an initial concentration of 0.5 mmol L(-1). However, only a small part of the substrate is mineralized. Two different experimental approaches (absolute and competition method) are adopted to estimate the second-order kinetic constants for the ozone attack at pH=6.0, 7.0 and 8.0. A good agreement was observed between the two kinetic methods adopted. The identification of main intermediates, attempted by high-performance liquid chromatograph (HPLC)-MS technique, indicates that the oxidation of BZF develops through both the hydroxylation of the aromatic ring and the attack of ozone on the unchlorinated aromatic one. The assessment of by-products biodegradability and acute toxicity demonstrates that ozonation is a suitable technique to improve the biodegradability and reduce the toxicity of waters containing BZF.

Toxic and genotoxic impact of fibrates and their photoproducts on non-target organisms.

Lipid regulators have been detected in effluents from sewage treatment plants and surface waters from humans via excretion. This study was designed to assess the ecotoxicity of fibrates, lipid regulating agents. The following compounds were investigated: Bezafibrate, Fenofibrate and Gemfibrozil and their derivatives obtained by solar simulator irradiation. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while SOS Chromotest and Ames test were utilized to detect the genotoxic potential of the investigated compounds. The photoproducts were identified by their physical features and for the first risk evaluation, the environmental impact of parental compounds was calculated by Measured Environmental Concentrations (MEC) using the available data from the literature regarding drug occurrence in the aquatic environment and the Predicted No Effect Concentrations (PNEC) based on our toxicity data. The results showed that acute toxicity was in the order of dozens of mg/L for all the trophic levels utilized in bioassays (bacteria, rotifers, crustaceans). Chronic exposure to these compounds caused inhibition of growth population on rotifers and crustaceans while the algae seemed to be slightly affected by this class of pharmaceuticals. Genotoxic and mutagenic effects were especially found for the Gemfibrozil photoproduct suggesting that also byproducts have to be considered in the environmental risk of drugs.

Role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in bezafibrate-induced hepatocarcinogenesis and cholestasis.

Prolonged administration of peroxisome proliferators to rodents typically leads to hepatocarcinogenesis. Peroxisome proliferator-activated receptor-alpha (PPARalpha) is required to mediate alterations in PPARalpha target gene expression, repress apoptosis, enhance replicative DNA synthesis, oxidative stress to DNA and hepatocarcinogenesis induced by the relatively specific PPARalpha agonist, Wy-14,643. Interestingly, administration of the less specific PPARalpha agonist, bezafibrate, leads to a modest induction of PPARalpha target genes in the absence of PPARalpha expression. In these studies, the role of PPARalpha in modulating hepatocarcinogenesis induced by long-term feeding of 0.5% bezafibrate was examined in wild-type (+/+) and PPARalpha-null (-/-) mice. The average liver weight was significantly higher in (+/+) and (-/-) mice fed bezafibrate than controls, but this effect was considerably less in (-/-) mice as compared with similarly treated (+/+) mice. Increased levels of mRNA encoding cell cycle regulatory proteins and DNA repair enzymes were found in (+/+) mice fed bezafibrate, and this effect was not found in (-/-) mice. In mice fed bezafibrate for 1 year, preneoplastic foci, adenomas and a hepatocellular carcinoma were found in (+/+) mice, while only a single microscopic adenoma was found in one (-/-) mouse. This effect was observed in both Sv/129 and C57BL/6N strains of mice, although only preneoplastic foci were observed in the latter strain. Interestingly, hepatic cholestasis was observed in 100% of the bezafibrate-fed (-/-) mice, and this was accompanied by significantly elevated hepatic expression of mRNA encoding bile salt export pump and lower expression of mRNA encoding cytochrome P450 7A1, consistent with enhanced activation of the bile acid receptor, farnesoid X receptor. Results from these studies demonstrate that the PPARalpha is required to mediate hepatocarcinogenesis induced by bezafibrate, and that PPARalpha protects against potential cholestasis.

The role of apoptotic regulators in metaplastic mucous cells.

Exposure of airways to environmental toxins or allergens induces proliferation of epithelial cells. Depending on the type of exposure, existing and newly formed cells can differentiate into mucus-producing cells resulting in mucous cell metaplasia (MCM). During recovery, the epithelium reduces the number of epithelial cells to return to the original state. Understanding the mechanisms involved in this resolution could be useful in deleting mucous cells and, thereby, mucous secretions. We have found that metaplastic mucous cells induced by exposure to ozone, endotoxin, cigarette smoke or allergens in epithelia of various regions of the airways express Bcl-2, a regulator of apoptosis, and neutrophils appear to be involved in its expression. The percentage of Bcl-2-positive mucous cells is decreased prior to the resolution of MCM. Furthermore, targeted reduction of Bcl-2 expression causes a dose-dependent reduction of epithelial mucous cells, suggesting that Bcl-2 is involved in maintaining metaplastic mucous cells. Horses with recurrent airway obstruction show an increased percentage of Bcl-2-positive mucous cells compared to their normal counterparts. These studies suggest that down-regulation of Bcl-2 expression may be useful to reduce mucous secretions in diseased subjects. The role of Bax in the reduction of MCM during prolonged exposure to allergen is also discussed.

Evaluation of kidney and liver subacute toxicity induced by Bezalip-Pravastatin-Lopid antihyperlipidaemic compounds in rats.

Renal and hepatic subacute toxicity induced by the antihyperlipidaemic drugs: Bezalip-Pravastatin and Lopid was investigated in rats using serum biochemical parameters. Toxicological evaluation was performed in serum samples following the administration of the therapeutic dose regimens of the compounds that were previously shown to be effective in inhibition of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase, the enzyme controlling the rate-limiting step in the synthesis of cholesterol, and acyl-CoA cholesterol acyl transferase (ACAT) which converts intracellular free cholesterol to cholesterol ester. Renal and hepatic subacute toxicity was evaluated by measuring enzyme activity or concentrations of: alanine aminotransferace, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, potassium, sodium, blood urea nitrogen, uric acid and creatinine. The use of the above serum biochemical parameters indicated that the overall toxicity impact of antihyperlipidaemic drugs was Bezalip = Pravastatin < Lopid. We have found that the Pravastatin--in contrast to the above antihyperlipidaemic drugs--only transiently affects the biochemical parameters associated with toxicity, but, it affects some of the biochemical parameters associated with hepatic and renal toxicity, up to a significantly lower extent than the antihyperlipidaemic drugs.

Lack of induction of hepatic DNA damage on long-term administration of peroxisome proliferators in male F-344 rats.

In order to evaluate the relationship between hydrogen peroxide (H2O2) generation and subsequent DNA damage caused by peroxisome proliferation, we examined DNA damage and changes in peroxisomal beta-oxidation activity in rat liver. Male F-344 rats were given orally clofibrate, bezafibrate or di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. In rats fed DEHP for 52 or 78 weeks hepatocarcinomas or neoplastic nodules were found. In rats treated for 2 weeks with peroxisome proliferators, peroxisomal beta-oxidation activity was increased 10-17 times over control levels. After long-term treatment (20-78 weeks), the level of peroxisomal beta-oxidation activity remained 3-13-times higher in each group. When single strand DNA breaks were measured by a DNA-alkaline elution technique, no increase in DNA damage was observed in livers from rats fed peroxisome proliferators for 2, 40 or 78 weeks. In rats bearing hepatocarcinomas induced by DEHP, the hepatic DNA showed significant breaks; the rate of DNA-alkaline elution was found to increase approximately 5-fold. No significant increase in hepatic lipid peroxide level was observed in each group. These results show that although prolonged treatment with peroxisome proliferators induces markedly peroxisomal beta-oxidation activity, the active oxygen species from peroxisomal beta-oxidation are not enough to give rise to significant DNA damage. Moreover, the change in the activity of peroxisomal beta-oxidation may not relate to hepatocarcinogenesis induced by peroxisome proliferators.

Long-term effects of peroxisome proliferators on the balance between hydrogen peroxide-generating and scavenging capacities in the liver of Fischer-344 rats.

In order to clarify whether peroxisomal hydrogen peroxide (H2O2) plays an important role in peroxisome proliferator-induced hepatocarcinogenesis, we examined the change in metabolism of peroxisomal H2O2 in vivo and in vitro using male Fischer-344 rats fed clofibrate, bezafibrate and di(2-ethylhexyl)phthalate (DEHP) for up to 78 weeks. Hepatic peroxisomal fatty acyl-CoA oxidase activity increased 12-20-fold after 2 or 4 weeks treatment; later this level gradually decreased toward controls, and at 78 weeks activity was 3-10-times of control. Although hepatic H2O2 levels were increased slightly by clofibrate, bezafibrate and DEHP, the changes did not correlate with the changes in peroxisomal fatty acyl-CoA oxidase activity. In isolated hepatocytes, the rate of leakage of peroxisomal H2O2 from peroxisomes into the cytosol and the hepatocellular H2O2 content was measured. The rate of leakage of peroxisomal H2O2 into cytosol increased 2.5-4-fold when peroxisomal beta-oxidation activity was induced by peroxisome proliferators, and the increases in this rate corresponded with changes in the peroxisomal beta-oxidation activity. In contrast, the hepatocellular H2O2 contents were not affected by induced peroxisomal beta-oxidation. These data show that H2O2 leaking from peroxisome into cytosol would be quickly decomposed, and thus peroxisomal H2O2 does not appear to play an important role in hepatocarcinogenesis by such an oxidative stress mechanism after the long-term treatment with peroxisome proliferators.

The comparative pharmacokinetics and gastric toxicity of bezafibrate and ciprofibrate in the rat.

1. The comparative gastric toxicology and pharmacokinetics of two phenoxyisobutyrate derivatives have been evaluated in the Fischer rat. 2. After oral administration of single daily doses for 7 days, the plasma elimination half-life for bezafibrate was rapid (t1/2 of 4-5 h) in comparison to ciprofibrate (t1/2 of 76 h). 3. The area under the plasma drug concentration versus time curve (AUC) 0-24 (micrograms.h/ml +/- SD) for bezafibrate (dose 125 mg/kg per day) was 1553 +/- 334, which was less than half the value of 3748 +/- 358 achieved by ciprofibrate (10 mg/kg per day) after 7 days. 4. Oral administration of ciprofibrate at 10 mg/kg every 48 h produced similar sustained plasma concentrations to those achieved by bezafibrate 125 mg/kg dosed every 12 h. The AUC 0-48 values (micrograms.h/ml +/- SD) achieved were 5124 +/- 450 for bezafibrate compared to 4207 +/- 240 for ciprofibrate. 5. In chronic oral multidose studies with ciprofibrate and bezafibrate, similar gastric toxicity (neuroendocrine cell hyperplasia) occurred in the rat when dose regimens were adjusted to compensate for the pharmacokinetic differences between these two drugs.