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204 Cytotoxic effects of malachite green in two human cell lines
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Poster Session P8. Food safety
once daily, 5 days per week for 13 weeks to groups of 10 male and 10 female F344/N rats and B6C3F1 mice at doses of 0, 37.5, 75, 150, 300, and 600 mg/kg. Additional groups of 10 male and 10 female rats were dosed similarly for 31 days and used for hematology and clinical chemistry measurements. All rats survived until the end of the study. One male and all female mice dosed with 600 mg/kg/day died or were sacrificed due to moribundity. Final mean body weights of male rats receiving estragole and final mean body weights of female rats and male and female mice dosed with 300 or 600 mg estragole/kg/day were lower than that of their respective control. Estragole also caused increases in liver weight of rats and mice. Estragole hematology and clinical chemistry related effects in rats included increased red blood cell count and decreased hemoglobin, hematocrit, mean corpuscular cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increased serum sorbitol dehydrogenase and alanine aminotransferase activities. Lesions related to estragole administration to rats included an increased incidence of lesions in the liver, salivary glands, glandular stomach, lung, nose, testes, and kidney. Treatment related lesions were observed in the liver, nose and the glandular stomach of mice. 202
METABOLISM AND CYTOTOXIC MECHANISMS OF SALICYLALDEHYDE IN ISOLATED RAT HEPATOCYTES
Hossein Niknahad 1 , Peter J. O’Brien 2 . 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, Iran, 71345; 2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 2S2 Salicylaldehyde (SA) is an aromatic aldehyde present in spices such as vanilla or cinnamon used as food additives and preservatives. In the present study SA was found to cause cytotoxicity towards freshly isolated rat hepatocytes with an LD50 of 0.6 mM for 2 hours. The events before plasma membrane disruption were immediate inhibition of mitochondrial respiration followed by depletion of ATP content of the cells. GSH content of hepatocytes was also partially depleted before cytotoxicity occurred. On the other hand, depleting GSH content of hepatocytes before the addition of SA increased the susceptibility of hepatocytes to SA and caused stronger inhibition of mitochondrial respiration. Increasing hepatocyte NADH levels with betahydroxybutyrate, xylitol, ethanol or lactate prevented SA-induced cytotoxicity while oxidizing NADH with acetoacetate increased its toxicity. The ATP generators fructose or dihydroxyacetone and the mitochondrial permeability transition pore inhibitor cyclosporin also prevented SA cytotoxicity, suggesting that SA is a mitochondrial toxin. Unlike other aldehydes, SA did not increase lipid peroxidation in hepatocytes, although induced some ROS formation. SA was not a good substrate for microsomal aldehyde dehydrogenase, which is important in the detoxification of aromatic aldehydes; however, SA was oxidatively detoxified by mitochondrial aldehyde dehydrogenase and inhibiting this enzyme by cyanamide or citral increased SA-induced cytotoxicity and reduced its LD50 to about 0.2 mM. On the other hand, SA was a good substrate for alcohol dehydrogenase, and salicyl alcohol formed was not cytotoxic even at a concentration nearly 10-fold higher. Inhibiting ADH with methylpyrazole increased SA cytotoxicity. SA was much more toxic than other aromatic aldehydes probably because it was poorly metabolized by aldehyde dehydrogenases while the aldehydes that were good substrates for these enzymes were less toxic than SA. This suggests that unmetabolized aldehydes could bind to critical intracellular targets, e.g. proteins, resulting in cytotoxicity. SA could also form a Schiff base with protein e-amino groups such as cytochrome c that can result in polymerization of cytochrome c and prevent its function.
203
ASSESSMENT OF EXPOSURE TO FOOD CHEMICALS, IN ROMANIA, 2001–2002
Carmen Hura 1 , I. Palamaru 1 , B.A. Hura 2 . 1 Food Toxicology Laboratory, Institute of Public Health, Iassy, Romania, 2 Electrotechnic Faculty, Technical University “Gh. Asachi” Iassy, Romania Ever since humans have become aware that health is inseparably linked to an impact and healthy environment, the control and reduction of pollution have become the focus of world wide concern. Investigations on possible health and environmental hazards involved have led many industrial countries to restrict or ban the use of chemicals (pesticides, heavy metals) and enforce the tolerance levels for the residues in food and feeds. The aim of the study was to investigate the variation of some chemical pollutants with cancer risk (nitrate/nitrite, heavy metals, pesticides residues) in some food (vegetables, meat, milk, fish, daily diets) from the Romania area, in 2001- 2002 period. The concentration of the nitrates/nitrites and the heavy metals (Cu, Cd, Pb, Mn, Zn, Ni) were investigated in 5386 food samples (vegetables (1364 samples), meat and meat products (2743 samples), milk and dairy products (952 samples), fish (55 samples), total diets (327 samples). The food were harvested from the Romania area (32 districts), in 2001- 2002 period. In the milk, vegetables, meat and total diets were analysed the metals (Cd, Pb, Zn, Cu, Ni, Mn, Fe) by absorption spectrophotometric method. The nitrate/nitrite were determined by colorimetric method and the pesticides residues by gas-chromatography. In all analysed samples these chemical pollutants were found. Generally, a wide variation between in individual samples were observed. Nitrate/nitrite contents were, generally, in normal limits and the total diets contained quantities below acceptable daily intake. The analysis of results obtained showed that in food was found the heavy metals in varied concentrations but in the admissible limits. The results give emphasis that the pesticide residues are present in all food analyzed. The determinations of chemical pollutants in food are important in environmental monitoring for the prevention, control and reduction of pollution as well as for occupational health, legal, decisions and epidemiological studies. 204
CYTOTOXIC EFFECTS OF MALACHITE GREEN IN TWO HUMAN CELL LINES
I. De Angelis 1 , A. Giuliano Albo 2 , C. Nebbia 2 , A. Stammati 1 , F. Zampaglioni 1 , M. Dacasto 2 . 1 Dipartimento Ambiente e prevenzione primaria, Istituto Superiore di Sanità, Roma, Italia. 2 Dipartimento di Patologia Animale, sezione di Farmacologia e Tossicologia, Università di Torino, Italia The triphenylmethane dye malachite green (MG) is still illegally used in aquaculture as a fungicide on larvae and juvenile fish. MG is rapidly absorbed, metabolised to its reduced derivative leucobase leucomalachite green (LMG) and then excreted. Apart from the LMG residues in muscular tissues of treated animals, MG has been reported to be a tumour promoter both in vitro and in vivo. The aim of this study was to assess the in vitro toxicity of MG. Two human cell lines were used: Hep-2 cells, derived from a human larynx carcinoma and Caco-2 cells, obtained from a human colon carcinoma. A reduction of cell viability, measured as NRU uptake and TPC content, was observed in Hep-2 cells treated for 24 hrs with different concentrations of MG (0.26–3.92 µM); the IC50 values obtained were 2.14 and 2.22 µM, respectively. Besides, MG reduced the proliferation capability, measured as CFA, of Hep-2 cells treated for 24 hr with MG and then sub-cultured in absence of the compound;. the relative IC50 value was 2.04 µM. As regards the Caco-2 cell model, dose-related increasing percentages of cytotoxicity, measured by NRU, LDH leakage and MTT assays, were observed in cells exposed for 24 hrs to different MG concentrations (0.1–100 µM); the IC50 values were 13.8µM, 18.4 µM and 16.2 µM for NRU, LDH leakage and MTT, respectively. In conclusion, MG was demonstrated to be cytotoxic to both Hep-2 and Caco-2 cells, and the former seems to be more sensitive to the dye toxic effect. The assessment of LMG cytotoxicity is actually under investigation. Supported by grants from Ricerca Finalizzata Regione Piemonte.
Poster Session P8. Food safety
once daily, 5 days per week for 13 weeks to groups of 10 male and 10 female F344/N rats and B6C3F1 mice at doses of 0, 37.5, 75, 150, 300, and 600 mg/kg. Additional groups of 10 male and 10 female rats were dosed similarly for 31 days and used for hematology and clinical chemistry measurements. All rats survived until the end of the study. One male and all female mice dosed with 600 mg/kg/day died or were sacrificed due to moribundity. Final mean body weights of male rats receiving estragole and final mean body weights of female rats and male and female mice dosed with 300 or 600 mg estragole/kg/day were lower than that of their respective control. Estragole also caused increases in liver weight of rats and mice. Estragole hematology and clinical chemistry related effects in rats included increased red blood cell count and decreased hemoglobin, hematocrit, mean corpuscular cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increased serum sorbitol dehydrogenase and alanine aminotransferase activities. Lesions related to estragole administration to rats included an increased incidence of lesions in the liver, salivary glands, glandular stomach, lung, nose, testes, and kidney. Treatment related lesions were observed in the liver, nose and the glandular stomach of mice. 202
METABOLISM AND CYTOTOXIC MECHANISMS OF SALICYLALDEHYDE IN ISOLATED RAT HEPATOCYTES
Hossein Niknahad 1 , Peter J. O’Brien 2 . 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, Iran, 71345; 2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 2S2 Salicylaldehyde (SA) is an aromatic aldehyde present in spices such as vanilla or cinnamon used as food additives and preservatives. In the present study SA was found to cause cytotoxicity towards freshly isolated rat hepatocytes with an LD50 of 0.6 mM for 2 hours. The events before plasma membrane disruption were immediate inhibition of mitochondrial respiration followed by depletion of ATP content of the cells. GSH content of hepatocytes was also partially depleted before cytotoxicity occurred. On the other hand, depleting GSH content of hepatocytes before the addition of SA increased the susceptibility of hepatocytes to SA and caused stronger inhibition of mitochondrial respiration. Increasing hepatocyte NADH levels with betahydroxybutyrate, xylitol, ethanol or lactate prevented SA-induced cytotoxicity while oxidizing NADH with acetoacetate increased its toxicity. The ATP generators fructose or dihydroxyacetone and the mitochondrial permeability transition pore inhibitor cyclosporin also prevented SA cytotoxicity, suggesting that SA is a mitochondrial toxin. Unlike other aldehydes, SA did not increase lipid peroxidation in hepatocytes, although induced some ROS formation. SA was not a good substrate for microsomal aldehyde dehydrogenase, which is important in the detoxification of aromatic aldehydes; however, SA was oxidatively detoxified by mitochondrial aldehyde dehydrogenase and inhibiting this enzyme by cyanamide or citral increased SA-induced cytotoxicity and reduced its LD50 to about 0.2 mM. On the other hand, SA was a good substrate for alcohol dehydrogenase, and salicyl alcohol formed was not cytotoxic even at a concentration nearly 10-fold higher. Inhibiting ADH with methylpyrazole increased SA cytotoxicity. SA was much more toxic than other aromatic aldehydes probably because it was poorly metabolized by aldehyde dehydrogenases while the aldehydes that were good substrates for these enzymes were less toxic than SA. This suggests that unmetabolized aldehydes could bind to critical intracellular targets, e.g. proteins, resulting in cytotoxicity. SA could also form a Schiff base with protein e-amino groups such as cytochrome c that can result in polymerization of cytochrome c and prevent its function.
203
ASSESSMENT OF EXPOSURE TO FOOD CHEMICALS, IN ROMANIA, 2001–2002
Carmen Hura 1 , I. Palamaru 1 , B.A. Hura 2 . 1 Food Toxicology Laboratory, Institute of Public Health, Iassy, Romania, 2 Electrotechnic Faculty, Technical University “Gh. Asachi” Iassy, Romania Ever since humans have become aware that health is inseparably linked to an impact and healthy environment, the control and reduction of pollution have become the focus of world wide concern. Investigations on possible health and environmental hazards involved have led many industrial countries to restrict or ban the use of chemicals (pesticides, heavy metals) and enforce the tolerance levels for the residues in food and feeds. The aim of the study was to investigate the variation of some chemical pollutants with cancer risk (nitrate/nitrite, heavy metals, pesticides residues) in some food (vegetables, meat, milk, fish, daily diets) from the Romania area, in 2001- 2002 period. The concentration of the nitrates/nitrites and the heavy metals (Cu, Cd, Pb, Mn, Zn, Ni) were investigated in 5386 food samples (vegetables (1364 samples), meat and meat products (2743 samples), milk and dairy products (952 samples), fish (55 samples), total diets (327 samples). The food were harvested from the Romania area (32 districts), in 2001- 2002 period. In the milk, vegetables, meat and total diets were analysed the metals (Cd, Pb, Zn, Cu, Ni, Mn, Fe) by absorption spectrophotometric method. The nitrate/nitrite were determined by colorimetric method and the pesticides residues by gas-chromatography. In all analysed samples these chemical pollutants were found. Generally, a wide variation between in individual samples were observed. Nitrate/nitrite contents were, generally, in normal limits and the total diets contained quantities below acceptable daily intake. The analysis of results obtained showed that in food was found the heavy metals in varied concentrations but in the admissible limits. The results give emphasis that the pesticide residues are present in all food analyzed. The determinations of chemical pollutants in food are important in environmental monitoring for the prevention, control and reduction of pollution as well as for occupational health, legal, decisions and epidemiological studies. 204
CYTOTOXIC EFFECTS OF MALACHITE GREEN IN TWO HUMAN CELL LINES
I. De Angelis 1 , A. Giuliano Albo 2 , C. Nebbia 2 , A. Stammati 1 , F. Zampaglioni 1 , M. Dacasto 2 . 1 Dipartimento Ambiente e prevenzione primaria, Istituto Superiore di Sanità, Roma, Italia. 2 Dipartimento di Patologia Animale, sezione di Farmacologia e Tossicologia, Università di Torino, Italia The triphenylmethane dye malachite green (MG) is still illegally used in aquaculture as a fungicide on larvae and juvenile fish. MG is rapidly absorbed, metabolised to its reduced derivative leucobase leucomalachite green (LMG) and then excreted. Apart from the LMG residues in muscular tissues of treated animals, MG has been reported to be a tumour promoter both in vitro and in vivo. The aim of this study was to assess the in vitro toxicity of MG. Two human cell lines were used: Hep-2 cells, derived from a human larynx carcinoma and Caco-2 cells, obtained from a human colon carcinoma. A reduction of cell viability, measured as NRU uptake and TPC content, was observed in Hep-2 cells treated for 24 hrs with different concentrations of MG (0.26–3.92 µM); the IC50 values obtained were 2.14 and 2.22 µM, respectively. Besides, MG reduced the proliferation capability, measured as CFA, of Hep-2 cells treated for 24 hr with MG and then sub-cultured in absence of the compound;. the relative IC50 value was 2.04 µM. As regards the Caco-2 cell model, dose-related increasing percentages of cytotoxicity, measured by NRU, LDH leakage and MTT assays, were observed in cells exposed for 24 hrs to different MG concentrations (0.1–100 µM); the IC50 values were 13.8µM, 18.4 µM and 16.2 µM for NRU, LDH leakage and MTT, respectively. In conclusion, MG was demonstrated to be cytotoxic to both Hep-2 and Caco-2 cells, and the former seems to be more sensitive to the dye toxic effect. The assessment of LMG cytotoxicity is actually under investigation. Supported by grants from Ricerca Finalizzata Regione Piemonte.