Effects of methylmercury contained in a diet mimicking the Wayana Amerindians contamination through fish consumption: mercury accumulation, metallothionein induction, gene expression variations, and role of the chemokine CCL2.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg(2+) has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2(-/-) mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2(-/-) mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells.

The chemokine CCL2 protects against methylmercury neurotoxicity.

Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Deleterious effects in mice of fish-associated methylmercury contained in a diet mimicking the Western populations' average fish consumption.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. Only a few contradictory epidemiological studies are currently available examining the impact of fish consumption on human populations. In the present study, we wanted to address whether a diet mimicking the fish consumption of Western populations could result in observable adverse effects in mice, and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of MeHg, if any. In Europe and the United States, fish consumption varies widely between countries, from 11 to 100 g fish/day. A mid-range value of 25 g fish/day corresponds to a fish contribution to the total diet of 1.25% on a dry weight basis. We decided to supplement a vegetarian-based mouse diet with 1.25% of lyophilized salmon flesh (SAL diet), or 1.25% of a blend of lyophilized cod, tuna, and swordfish (CTS diet). Total mercury contents were 1.15±0.15, 2.3±0.1 and 35.75±0.15 ng Hg/g of food pellets for the control, SAL and CTS diets, respectively. After two months feeding, the CTS diet resulted in significant observable effects as compared to the control and SAL diets, encompassing decreased body growth, altered behavioral performance and increased anxiety level, modification of mitochondrial respiratory protein subunit concentrations in kidney and brain structures, modified gene expression patterns in kidneys, liver and muscles, and a decrease of dopamine concentrations in the hypothalamus and striatum. Our findings have health implications, firstly because 1.25% of CTS flesh in the diet corresponds to an average exposure to MeHg below the WHO provisory tolerable weekly intake (PTWI) (1.6 µg MeHg/kg of body weight/week), and secondly because many people in Western populations, among them women of child-bearing age, are exceeding the PTWI value (for instance, 35% of the French population inhabiting the Atlantic and Mediterranean coasts).

Feeding mice with diets containing mercury-contaminated fish flesh from French Guiana: a model for the mercurial intoxication of the Wayana Amerindians.

BACKGROUND: In 2005, 84% of Wayana Amerindians living in the upper marshes of the Maroni River in French Guiana presented a hair mercury concentration exceeding the limit set up by the World Health Organization (10 microg/g). To determine whether this mercurial contamination was harmful, mice have been fed diets prepared by incorporation of mercury-polluted fish from French Guiana. METHODS: Four diets containing 0, 0.1, 1, and 7.5% fish flesh, representing 0, 5, 62, and 520 ng methylmercury per g, respectively, were given to four groups of mice for a month. The lowest fish regimen led to a mercurial contamination pressure of 1 ng mercury per day per g of body weight, which is precisely that affecting the Wayana Amerindians. RESULTS: The expression of several genes was modified with mercury intoxication in liver, kidneys, and hippocampus, even at the lowest tested fish regimen. A net genetic response could be observed for mercury concentrations accumulated within tissues as weak as 0.15 ppm in the liver, 1.4 ppm in the kidneys, and 0.4 ppm in the hippocampus. This last value is in the range of the mercury concentrations found in the brains of chronically exposed patients in the Minamata region or in brains from heavy fish consumers. Mitochondrial respiratory rates showed a 35-40% decrease in respiration for the three contaminated mice groups. In the muscles of mice fed the lightest fish-containing diet, cytochrome c oxidase activity was decreased to 45% of that of the control muscles. When mice behavior was assessed in a cross maze, those fed the lowest and mid-level fish-containing diets developed higher anxiety state behaviors compared to mice fed with control diet. CONCLUSION: We conclude that a vegetarian diet containing as little as 0.1% of mercury-contaminated fish is able to trigger in mice, after only one month of exposure, disorders presenting all the hallmarks of mercurial contamination.

Alterations of the bioenergetics systems of the cell in acute and chronic myocardial ischemia.

The aim of the works presented here is to analyze the alterations induced by acute ischemia-reperfusion and chronic ischemia on mitochondrial function, in relation to alterations on heart function. Parameters of mitochondrial function were assessed on skinned fibers coming from isolated perfused rat hearts. The effects of chronic ischemia were studied on a rat model of left descending coronary artery stenosis. Two key events observed after acute ischemia-reperfusion and chronic ischemia are the decrease (or the loss) of the stimulatory effect of creatine and the alteration of outer mitochondrial permeability to cytochrome c and ADP. Taken together, these effects indicate the alteration of the intermembrane space architecture leading to the loss of intracellular adenine nucleotides compartmentation and possibly of functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase. These alterations result in the impairment of intracellular energy transfer (channeling) from mitochondria to ATP-utilizing sites located in the cytosol. This may play a significant role in ischemic injury and alterations in heart function. We show that these effects were prevented by effective cardioprotective strategies like ischemic preconditioning or pharmacological preconditioning by perfusion of mitochondrial ATP-sensitive potassium channel openers. We hypothesize that an open mitochondrial ATP-sensitive potassium channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP.

Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart.

Diazoxide opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK(ATP) channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK(ATP) channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK(ATP) channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.

Histochemical evidence for pathways insulin cells use to oxidize glycolysis-derived NADH.

The activity of lactate dehydrogenase is known to be low in the pancreatic beta cell, and the activity of the mitochondrial glycerol phosphate dehydrogenase (mGPD), the key enzyme of the glycerol phosphate shuttle, is known to be high in this cell. Lactate dehydrogenase was demonstrated histochemically in insulin positive cells of the rat pancreas, and its activity was semiquantified densitometrically; activity in these cells was estimated to be about 8% of that in the surrounding acinar tissue. mGPD histochemical activity was extremely high in cells exhibiting insulin immunofluorescence, while activity in surrounding pancreas tissue was negligible. When the activity was measured in situ at a physiologic concentration of substrate, this enzyme was inactive in the absence of free calcium. These results are consistent with the idea that glucose, the most potent physiologic insulin secretagogue, stimulates insulin secretion via aerobic glycolysis. If glycolysis-derived NADH, instead of being reoxidized in a mitochondrial hydrogen shuttle, is reoxidized to NAD by pyruvate via the reaction catalyzed by lactate dehydrogenase with the resulting formation of lactate, there would be little or no pyruvate available for mitochondrial metabolism. Consequently adenosine triphosphate formation would be about 5% to 7% of that formed by the complete combustion of glucose to carbon dioxide via mitochondrial metabolism. The low lactate dehydrogenase and high mGPD emphasize the importance of mitochondrial hydrogen shuttles for reoxidation of glycolysis-derived NADH in insulin secretion.

Alteration of mitochondrial function in a model of chronic ischemia in vivo in rat heart.

The aim of this study was to investigate mitochondrial alterations in an animal model of chronic myocardial ischemia in rats obtained by surgical constriction of the left coronary artery. Resting coronary blood flow was measured using the fluorescent microsphere technique. Contractile function, defined by rate-pressure product, and myocardial oxygen consumption were measured in a Langendorff preparation. The mitochondrial function was evaluated on permeabilized skinned fibers. Three weeks after surgery, ischemic hearts showed a significant decrease in coronary blood flow compared with sham. Hemodynamic measurements showed a significant systolic and diastolic dysfunction. Alterations in mitochondrial function in ischemic hearts were mainly characterized by a significant decrease in the maximal velocity and apparent half-saturation constant for ADP, loss of the stimulatory effect of creatine, and a stimulatory effect of exogenous cytochrome c. These functional alterations were supported by structural alterations characterized by mitochondrial clustering and swelling associated with membrane rupture. We conclude that the alterations in systolic function after chronic ischemia are supported by severe modifications of mitochondrial structure and function.