Generation of oxyradicals (O2. and H2O2), mitochondrial activity and induction of apoptosis of PBMC of Cyprinus carpio carpio treated in vivo with halomethanes and with recombinant HSP60 kDa and with LPS of Klebsiella pneumoniae.

Halomethanes (HM) can be immunotoxic in mammals; however, in the fish immune system HM effects are unknown. In the current study, we evaluated the mitochondrial activity (MA) by MTT, induction of apoptosis by SubG0 technique and quantified serum ROS concentration (O2. and H2O2) and ROS production in PBMC of Cyprinus carpio carpio treated i.p. with CH2Cl2, CHCl3 and BrCHCl2 (0.004-40.0 mg/kg) for 96 h. Positive controls were recombinant heat shock protein of 60 kDa (rHSP60 kDa) of Klebsiella pneumoniae and its LPS. In addition, for in vitro PBMC cultures, two culture media and two sources of sera were tested. Both positive controls increased the MA more than 4-fold as well as the production of O2. (26-fold) and H2O2 (5-fold) compared to their controls. HM induced different effects on MA, ROS production and an induction of apoptosis, depending on the chlorination patterns and the dose; however, a systemic damage prevails. To fish treated with CH2Cl2, the apoptosis was related with serum ROS concentration and with MA. In contrast, in fish dosed with CHCl3 relationships were not found, deducing a systemic damage. However, in fish treated with BrCHCl2, serum O2. concentration and in vitro ROS generation performed by PBMC were involved in the induction of apoptosis of these cells but not with MA suggesting also immunotoxic effects. The current study demonstrated that HMs are immunomodulators increasing an acute inflammatory response and that rHSP60kDA of K. pneumoniae and its LPS are appropriate antigens to assess the immune response of C. c. carpio.

The relationship between the bioactivation and detoxification of diazinon and chlorpyrifos, and the inhibition of acetylcholinesterase activity in Chirostoma jordani from three lakes with low to high organophosphate pesticides contamination.

In fish, a number of studies have linked acetylcholinesterase (AChE) inhibition with exposure to organophosphate pesticides (OPs); however, evidence suggests the need to study aspects related to the bioactivation and detoxification of OPs, since their neurotoxicity is dependent on these processes. Thus, the study aim was to examine the relations between chlorpyrifos (CPF) and diazinon (DZN) bioactivation by hepatic CYP450 izoenzymes (CYP 2B6, CYP 2C19, CYP 3A4) and detoxification by aryl esterases and oxonases with brain and muscle AChE activity in Chirostoma jordani from three lakes with low to high OPs contamination in water and sediments. We found two patterns of bioactivation in vitro: (i) in fish from a lake with high CPF pollution, the main isoenzymes involved in this process were CYP 2C19>CYP 2B6>CYP 3A4, and (ii) in fish captured in a lake with a high concentration of DZN, the isoenzymes were CYP 3A4>CYP 2C19>CYP 2B6. Bioactivation is shown in this study to be fundamental in brain and muscle AChE inhibition in vivo. The rate of bioactivation of CPF was lower than for DZN. CPF bioactivation was accompanied by reduced detoxification and higher neurotoxicity, which was inversely dependent on the environmental contamination of CPF. Detoxification was also inversely correlated with environmental contamination by CPF, and was higher with diazoxon than chlorpyrifos-oxon. Oxonases were the most relevant enzymes involved in detoxification. The current findings suggest a series of strategies between the bioactivation and detoxification of OPs that allowed the survival of C. jordani despite of OPs pollution levels.

Halomethane-induced cytotoxicity and cell proliferation in human lung MRC-5 fibroblasts and NL20-TA epithelial cells.

Halomethanes (HMs) can be formed during the chlorination process to obtain drinking water. In liver cells, HMs had been shown to be mutagenic and carcinogenic; however, their bioactivation by CYP 2E1 and GSTT1 is required. Although inhalation is the most common pathway of exposure, reports on the toxic effects induced by HMs in human lung are contradictory. The aim of this study was therefore to evaluate in vitro cytotoxicity and cell proliferation induced by CH(2)Cl(2), CHCl(3) and BrCHCl(2) in human lung NL20-TA epithelial cells and MRC-5 fibroblasts, and their relationship with CYP 2E1 and GSTT1 activity. High concentrations of these HMs induced cytotoxicity, particularly in cells treated with BrCHCl(2). Low concentrations of BrCHCl(2) stimulated hyperproliferation of fibroblasts, the most probable consequence of which is regenerative proliferation related to collagen induction. Fibroblasts exposed to BrCHCl(2) exhibited low levels of CYP 2E1 activity suggesting that released bromine is able to alter this activity by affecting the active site or auto regulating the activity itself. GSTT1 was up to ten times more active than CYP 2E1 in both cell lines, indicating that potential lung damage is due to formation of pro-carcinogens such as formaldehyde.

Implications of cytochrome 450 isoenzymes, aryl-esterase and oxonase activity in the inhibition of the acetylcholinesterase of Chirostoma jordani treated with phosphorothionate pesticides.

Organophosphate pesticides must be metabolized by cytochrome-P450 isoenzymes such CYP 2C19 as CYP 3A4 to induce neurotoxicity, but damage apparently depends on the activity of aryl esterases of the oxonase type that are involved in detoxication of these compounds. However, information on this subject is not available in fish. Chirostoma jordani has sustained significant population reductions, probably due to changes in land-use as well as pesticide impact; nevertheless, no specific studies demonstrating this are available. This study shows for the first time that the activity of cytochrome-P450 isoenzymes (CYP 2B6, CYP 2C19, CYP 3A4) in C. jordani is involved in diazinon and chlorpyrifos bioactivation. However, higher toxicity of chlorpyrifos cannot be explained solely because its bioactivation. Differences in toxicity between both pesticides are due to the activity of aryl esterases and oxonases that are responsible for oxon detoxication. Both hepatic enzymes metabolize diazoxon more efficiently than chlorpyrifos oxon. At lethal concentrations, detoxication is particularly important since mortality was lower with diazinon (LC50=1.5 µg/L) than with chlorpyrifos (LC50=0.17 µg/L). At sublethal levels, maximum acetylcholinesterase inhibition took place at 4h in both brain and muscle and was of lower magnitude in diazinon-treated fish. This is due to the higher affinity of both aryl esterases for diazoxon, which allows higher detoxication rates and therefore greater recovery of acetylcholinesterase activity.

Phase I enzyme induction in Girardinichthys viviparus, an endangered goodeid fish, exposed to water from native localities enriched with polychlorinated biphenyls.

The present study examines the induction of mixed-function oxidase (MFO) enzymes, including CYP content CYP1A (EROD) activity and alcohol dehydrogenase activity (ADH), in an endemic Mexican fish species, the black-fin goodeid Girardinichthys viviparus, exposed to the water of two localities, Lake Texcoco (LTX) and Lake Zumpango, and to the same matrices enriched in polychlorinated biphenyls (PCBs) to simulate the potential toxic effects of sublethal increases in these xenobiotics. Fishes of both sexes born in the laboratory were exposed for 1, 2, 4, 8, and 16 days. Water from the two types of localities of the black-fin goodeid contains MFO inducers. Of the two, the most contaminated is LTX water, which also contains PCBs. EROD activity was higher in all treatments with female compared with male fish. This suggests greater metabolic compromise in female fish as a response to damage caused by these xenobiotics. In this species, CYP induction displayed two patterns that were not always concurrent with higher CYP1A activity. In the enriched matrix system, biotransformation processes were notably altered. Increased ADH may indicate that this enzyme is involved in the biotransformation of PCBs and their metabolites, particularly in male fish, and provides at least a part of reductive power required by the MFO enzymes; however, specific studies are needed to clarify this point.

Estrogenic effects of polychlorinated biphenyls and relation to cytochrome P4501A activity in the endangered goodeid fish Ameca splendens.

The present study examines the relationships between cytochrome P4501A (CYP1A) activity and vitellogenin (VTG) induction in Ameca splendens elicited by a polychlorinated biphenyl (PCB) mixture. Ethoxyresorufin-O-deethylase (EROD) activity, mRNA levels of VTG, and VTG induction were evaluated in male and female fish exposed for 1, 2, 4, 8, and 16 d to a commercial PCB mixture. Polychlorinated biphenyls induced higher EROD in both sexes and this induction was higher in females than in males. Maximum EROD and VTG induction occurred on day 1 in females, while in males these maxima occurred on days 8 and 16. A correlation between EROD and VTG induction was found only in males (p<0.001), and VTG induction was also higher in males than in females (p<0.01). Exposure to PCBs elicited increases in VTG expression and induction over time in males, while in females these decreased at the end of the exposure period. Deficiencies in the feedback mechanisms of male A. splendens exposed in the wild to xenoestrogens such as PCBs have probably contributed to alter the sex ratio of wild populations of this species.