M. Lafaurie

Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species.

Cadmium poses a serious environmental threat in aquatic ecosystems but the mechanisms of its toxicity remain unclear. The purpose of this work was first to determine whether cadmium induced apoptosis in trout hepatocytes, second to determine whether or not reactive oxygen species (ROS) were involved in cadmium-induced apoptosis and genotoxicity. Hepatocytes exposed to increasing cadmium concentrations (in the range of 1-10 microM) showed a molecular hallmark of apoptosis which is the fragmentation of the nuclear DNA into oligonucleosomal-length fragments, resulting from an activation of endogenous endonucleases and recognized as a DNA ladder on conventional agarose gel electrophoresis. Exposure of hepatocytes to cadmium led clearly to the DEVD-dependent protease activation, acting upstream from the endonucleases and considered as central mediators of apoptosis. DNA strand breaks in cadmium-treated trout hepatocytes was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect DNA primary damage in individual cells. Simultaneous treatment of trout hepatocytes with cadmium and the nitroxide radical TEMPO used as a ROS scavenger, reduced significantly DNA fragmentation, DEVD-related protease activity and DNA strand breaks formation. These results lead to a working hypothesis that cadmium-induced apoptosis and DNA strand breaks in trout hepatocytes are partially triggered by the generation of ROS. Additional studies are required for proposing a mechanistic model of cadmium-induced apoptosis and genotoxicity in trout liver cells, in underlying the balance between DNA damage and cellular defence systems in fish.

Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea

Specimens of sea bass (Dicentrarchus labrax) were placed in cages for 1 month in spring and autumn at different locations in the Bay of Cannes (NW Mediterranean). Biochemical markers evaluated were: ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities in fish livers and acetylcholinesterase (AChE) activity in fish muscle. EROD and GST activities were higher in front of the outlet for the wastewater plant of Cannes and in the harbour than outside the marina. High EROD and GST activities may be induced by petrol hydrocarbons and/or polychlorinated biphenyls (PCBs). AChE was low in the muscles of the fish caged in the harbour compared with samples from the other cages. Low AChE activity could suggest the presence of organophosphorus and carbamate compounds in seawater from the harbour. Mussels (Mytilus galloprovincialis) were caged off Cannes for the same periods as the fishes. Heavy metal, metallothionein (MT) concentrations and lysosomal membrane stability were evaluated in the digestive gland of the mussels. Results show low heavy metal and MT concentrations, implying low metal concentrations in the surrounding waters. High lysosomal membrane stability revealed a good physiological status of these animals after caging. The whole set of data indicates that seawater in the Bay of Cannes appeared to be unpolluted as regards pollutants which may induce the measured biomarkers, except in restricted areas.

Metallothionein determination in the liver of the sea bass Dicentrarchus labrax treated with copper and B(a)P

Metallothioneins (MTs) were quantified in the liver of the sea bass Dicentrarchus labrax treated by intraperitoneal injection of copper (500 ng g−1), of benzo(a)-pyrene (20 μg g−1), and of copper (500 ng g−1) and benzo(a)pyrene (20 μg g−1). Two methods of determination of MTs were used: differential pulse polarography and spectrophotometry. Results showed a good correlation between the MT levels determined by both methods. When fishes were injected with copper, their MT levels were lower than in the case of controls, probably due to the toxic effect of copper whereas when fishes were injected with both pollutants there was a 2-fold increase in MT level, compared to controls. In the case of injection with BaP alone, hepatic MT level was even lower than that of controls or in the fishes treated with copper.

The nitroxide stable radical tempo prevents metal-induced inhibition of CYP1A1 expression and induction.

Heavy metals are known to provoke oxidative stress in fish liver cells. Because H2O2, OH*- and intracellular superoxide are involved in this oxidation, we investigated the effect of nitroxide radical, 2,2,6,6-tetramethylpiperidinyl-N-oxyl (abbreviated as TEMPO), a cell-permeable agent possessing antioxidant properties, on CYP1A expression in trout (Oncorhynchus mykiss) hepatocytes. 3-methylcholanthrene (3-MC) induced the CYP1A-related EROD activity. This induction was inhibited by concomitant exposure to Cd (II), Cu (II), Pb (II) or Zn (II). CYP1A mRNA levels were also reduced. Simultaneous treatment with 3-MC, a heavy metal and TEMPO suppressed both the inhibition of EROD activity and the decrease of CYP1A mRNA expression. These results suggest a working hypothesis that heavy metals produce multiple oxidative effects, including generation of hydroxyl radicals, which could down-regulate CYP1A1 expression. This metal-induced inhibition was prevented by TEMPO, which might protect trout hepatocytes by scavenging free radicals and thus preventing their inhibitory effects on CYP1A induction and expression.

Tissue-specific induction and inactivation of cytochrome P450 catalysing lauric acid hydroxylation in the sea bass, Dicentrarchus labrax.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

Molecular cloning of a CYP1A cDNA from the teleost fish Dicentrarchus labrax

A cDNA encoding for cytochrome P450 1A has been cloned in the marine teleost fish Dicentrarchus labrax. This fish, common in the Mediterranean, was chosen since it is considered a good sentinel species. Moreover, biomarkers of exposure to organic contaminants (such as EROD) are often measured in this species and make it possible to evaluate the quality of waters. For cloning purposes, RNAs were extracted from the liver of benzo[a]pyrene (BaP)-treated animals and used as template in degenerate RT-PCR. The cDNA product was cloned and used for the design of highly stringent primers that were utilized in Rapid Amplification of cDNA Ends (RACE) PCR. The cloned cDNA hybridizes with a 2.7 kb mRNA which is induced by treatment of the fish with BaP, a classical CYP1A inducer. The closest sequences found in data banks belong to fish CYP1A.

Toxic effects of several types of antifouling paints in human and rat hepatic or epidermal cells

Fouling is the successive development of marine organisms on immersed surfaces, a process which has heavy negative economic impacts. Several antifouling technologies, generally based on the leaching of biocides from painted surfaces, have been developed, but these biocides are toxic to the environment. Hence, we compared the toxicity of several currently used paint lixiviats in rat hepatocytes, human HepG2 and HaCaT cells. Acute toxicity was assessed by the Neutral Red and MTT assays. Chronic effect was tested using induction of the 7-ethoxyresorufin-O-deethylase (EROD) activity as a marker. Large variations were observed among the various cell types or the antifouling formulations, both in terms of IC50 values (from approximately 0.5 to approximately 10%, v/v) and EROD induction (from approximately 1 to 10-fold over control). These differences appear to be related to variable biocide (copper compounds, organotins, etc...) concentrations in the different paint formulations, or to the specific metabolic capabilities of the cell system used.

Expression and Induction of Cyp1A1 in Black Seabream (Spondyliosoma Cantharus) Hepatocyte Cultures: Effects of Heavy Metals

Abstract We developed a new method, which allowed black seabream hepatocytes to be maintained in primary-culture for several days. This method was shown to be suitable for studying sea fish CYPs expression and regulation following xenobiotic treatment. After isolation, hepatocytes were directly mixed with a type I collagen gel and culture medium, seeded in 6-well tissue culture plates (2.106 cells/35 mm well) and incubated under atmospheric air at 20°C. After 24 hrs, the cells were exposed for 1, 2 and 3 days to 3-methylcholanthrene (3MC), heavy metals (Cd(II), Cu(II), Pb(II), Zn(II), or 3MC with increasing concentrations of these metals. Cytotoxicity was assessed by the neutral red test: the sequence was Cd(II) > Cu(II) > Pb(II) > Zn (II) (EC50: 57.234, 544 and 722 μM respectively) and appeared to be correlated with the metal solubility. CYP1A1 expression was monitored by EROD activity as well as by Western and Northern blots. 3MC induced the CYP1A1-related EROD activity in a time-and dose-dependent mann...

Chapter 13 – EVALUATION OF VARIOUS BIOMARKERS IN THE WILD FISH SERRANUS CABRILLA COLLECTED IN THE NW MEDITERRANEAN SEA.

Relationships between chemical contaminant exposure and alterations in several biochemical processes in fish may allow the use of certain biochemical parameters as markers (commonly referred to as biomarkers) of exposure and early responses to chemical contaminants. The measurement of biochemical and physiological responses to chemical contaminants in fish may serve to improve the assessment of biologically significant exposure to toxic anthropogenic chemicals and enhance the ability to assess the risk for effects on the health and survival of contaminant-exposed populations. Many field studies demonstrated the interest of the measurement of cytochrome P450 dependent MFO (mixed function oxidases), and among them EROD (ethoxy resorufin O-deethylase) activity induced by polycyclic aromatic hydrocarbons (PAHs), in fish as exposure biomarkers in the aquatic environment. Other biomarkers may be measured in marine animals : glutathione S-transferase (GST) activity, acetylcholinesterase (AChE) activity. It has been demonstrated that GSTs may be induced by both PAHs and polychlorobiphenyls (PCBs). AChE activity is inhibited in the presence of pesticides such as organophosphorous compounds and carbamates. n nA cruise “BIOMAR” was performed in autumn 1995 along the coasts of the NW Mediterranean and Corsica to collect many specimens of the comber Serranus cabrilla. Three biomarkers (EROD, GST and AChE) were measured in this fish (in the liver for EROD and GST, and in the muscle for AChE) as well as heavy metal concentrations (Cd, Cu, Hg and Zn, determined in the gills). The statistical treatment of the data (Principal Component Analysis performed with564 data) allowed to discriminate polluted areas from clean areas. Areas such as Cortiou (which is the cove where the treated waste waters from Marseilles are discharged) and North Planier (located in an area exposed to chemical contaminants) were affected by multiple sources of pollution (since high EROD, GST activities, low AChE activities and elevated metal concentrations were observed), whereas, in sites of Corsica, the source of pollution appeared to be unique, since petroleum contamination could be detectable in some samples (high EROD values). The results suggest that the complementary use of several biomarkers facilitates the evaluation of the possible sources of contamination in fish collected from the natural environment.

ISOLATION OF CYTOCHROME P450 CDNAS (CYP1A1 AND CYP4T2) FROM THE SEA BASS (DICENTRARCHUS LABRAX) : TOOLS FOR BIOMONITORING SEA POLLUTION.

Abstract Cytochrome P450 are key detoxification enzymes which are potentially induced by xenobiotics such as environmental contaminants. Thus, induction features of some cytochrome P450 are used as biomarkers. Many studies use enzyme activities to detect this induction, but this can also be monitored by protein or mRNA quantitation. There are about 80 different cytochrome P450 genes in vertebrates, but only few sequences are available in fish. Family 1, 2 and 3 are now well studied, but mostly in trout. We isolated CYP1A and CYP4T2 sequences in the sea bass Dicentrarchus labrax, which is considered as an excellent sentinel species in the Mediterranean sea. CYP1A isoform is induced by PAHs, PCBs, and dioxins, and some CYP4 isoforms are known to be induced by phthalate esters plasticizers and chlorinated aryl phenoxy herbicides. We characterize the induction of the genes by these environmental contaminants in several organs. This study gives some knowledge about cytochrome P450 gene regulation in the sea bass, which is essential for further use of these genes as biomarkers. Quantitation of mRNA could be used as additional tools to report CYP1A and CYP4T2 induction, and could lead to the development of efficient biomarkers for these class of marine pollutants.