- Email: [email protected]
Induction of metallothionein in cultured human proximal tubular cells can protect against cisplatin induced nephrotoxicity
Poster Session 1C. In Vitro Mechanism the five monoesters inhibited GJIC in hamster or human hepatocytes and in a human liver cell line. This GJIC assay has good cancer predictive potential for phthalates (Toxicologist 15: #1469, 1995). In conclusion, these results indicate that some phthalate monoesters can inhibit GJIC in rat and mouse hepatocytes but not all monoesters show equivalence to MEHP. Moreover, the qualitative species differences (i.e., humans and hamsters insensitive to GJIC inhibition) and other data suggest that phthalate esters pose a negligible (liver) cancer risk for man.
35
of protection. Transforming growth factor-fl and epidermal growth factor, although induced MT did not seem to confer the same level of cytoprotection against cisplatin-induced toxicity in HPT cells. Work supported by the Wellcome Trust (Ref: 038369/Z/93/Z/ PMG/RB).
Keywords: metallothionein; proximal tubules; cisplatin; nephrotoxicity; human; in vitro
P1C-1301 MORPHOLOGICAL ALTERATIONS INDUCED BY IP1C-128
CADMIUM, MERCURY, ZINC AND BISMUTH ON CULTURED HUMAN PROXIMAL TUBULAR CELLS
1 VALUE OF LIVER TISSUE DOSlMETRY DATA TO
I
DESIGN AND INTERPRET IN VITRO (HEPATOCYTE) GAP JUNCTION STUDIES WITH PEROXISOMAL PROLIFERATING AGENTS
A.W. Lington 1, G.H. Kalimi 2, J.E. Klaunig 2, A.I. Nikiforov * 1
i Exxon Biomedical Sciences, Inc.; 2 Indiana University Peroxisomal proliferating agents (PPA), such as trichloroethylene (TCE) and di-2-ethylhexyl phthalate (DEHP), are non-genotoxic liver carcinogens in mice (DEHP, TCE) and/or rats (DEHP). In vitro studies employing hepatocyte cultures are being used for PPA with a focus on inhibition of gap junctional intercellular communication (GJIC). Inhibition of GJIC has been proposed as a promising assay for tumor promoters. DEHP is readily hydrolyzed to the monoester (MEHP) in vivo and MEHP is the main chemical found in rodent liver. DEHP (1000/zM) failed to inhibit GJIC inhibition in rat/mouse hepatocytes, most likely due to poor hydrolysis to MEHP, while MEHP (150-500 /zM) inhibited GJIC in rat/mouse and was readily metabolized in this system. TCE is extensively metabolized to trichloroacetic acid (TCA) in mice but not rats (in vivo, hepatocytes). TCE and TCA inhibited GJIC in mouse hepatocytes but not in rat hepatocytes. In conclusion, metabolic and tissue dosimetry data on PPA can assist in the design and data interpretation on whether GJIC effects in vitro are due to parent chemical or metabolites and also provide a sound basis for species differences.
I P 1I C - 1 2 9 I
INDUCTION OF METALLOTHIONEIN IN CULTURED HUMAN PROXIMAL TUBULAR CELLS CAN PROTECT AGAINST CISPLATIN INDUCED NEPHROTOXlCITY
Vicente RodiUa *, Jonathan P. Little, Naheed Afzal, Adrian T. Miles, Gabrielle M. Hawksworth. University of Aberdeen, Departments of
Medicine and Therapeutics and Biomedical Sciences, Foresterhill, Aberdeen AB9 2ZD, Scotland Metallothioneins (MTs) are low molecular weight metal binding proteins characterized by their high cysteine content. MTs are thought to play a role in the protection against heavy metal toxicity, may act as free radical scavengers and maybe be involved in the homeostasis of essential metals such as copper and zinc. These proteins are not only inducible by metals but also by exposure to a wide range of physical and chemical agents including stress, cytokines, growth factors, hormones and corticosteroids. Cisplatin is one of the most widely used anticancer agents whose dose limiting factor is its nephrotoxicity. Cisplatin produces DNA damage, generates free radicals and induces lipid peroxidation. In this study we investigated the possible effects of chemically-induced metallothionein as a prospective protective protein in cisplatin induced nephrotoxicity in man using cultured human proximal tubular (HPT) cells. The results of our studies demonstrate that dexamethasone, interleukin-6, prostaglandin Et, ZnCI2 and Bi(NO3)3 all induce metallothionein as demonstrated using immunocytochemistry. Maximal MT induction was achieved 48 hours after exposure to the chemical. The cytoprotection exerted by this induced metallothionein on cisplatin-treated HPT cultures was evaluated using the MTT assay and resulted in varying degrees
Vicente Rodilla *, Adrian T. Miles, Deborah Marshall ], Gabrielle M. Hawksworth. University of Aberdeen, Departments of Medicine
and Therapeutics and Biomedical Sciences, Foresterhill, Aberdeen AB9 2ZD, Scotland; I Electron Microscopy Unit, Department of Medical Microbiology, Foresterhill, Aberdeen AB9 2ZD, Scotland The kidney in particular the proximal convoluted tubule is a major target for the toxic effects of various metals. In the present study we have investigated the toxicity of several inorganic metal compounds (CdCI2, HgC12 ZnCI2 and Bi(NO3)3) upon cultured human proximal tubular (HPT) cells. These cells were isolated from the cortex of nephrectomy specimens and purified by centrifugation in Percoll. The toxic effects of various concentrations of the metals were determined using the MTT assay. Mercury was the most toxic compound, followed by cadmium, zinc, and bismuth, in that order. The morphological alterations caused by exposure of the cells to the metals were determined using optical and electron microscopy (SEM). Although exposure to the metals caused varying effects, the treated cells acquired a spherical morphology. Exposure to mercury caused other morphological alterations such as elongation and irregular distortion of the cells. Additional SEM observations revealed a number of alterations at membrane level such as blisters and other protuberances including small blebs. In the case of cells exposed to CdCI2 and ZnC12 the integrity of the cell membrane was destroyed and holes and small craters appeared, eventually leading to the destruction of the cell. This work has been supported by the Wellcome Trust (Refs: 038369/Z/93/Z/PMG/RB and 046936)
Keywords: heavy metal toxicity; proximal tubules; human; in vitro; SEM I
I P1 C-1 31 I I
NITRIC OXIDE SYNTHASE GENE INDUCTION BY KILLED PROPIONIBACTERIUM A VIDUM IN RAT KUPFFER AND ENDOTHELIAL CELLS IN VITRO
Mounir Dhouib * l, Cathy Royer 2, Jean-Louis Gendrault 2, Alain Lugnier i. i University Louis Pasteur, Faculty of Pharmacy,
Department of Toxicology and Faculty of Medicine, Strasbourg, France; 2 University Louis Pasteur, Faculty of Pharmacy, Laboratory of Virology and INSERM U74, Strasbourg, France Macrophages protect against bacterial infections by the production of reactive molecules such as nitric oxide (NO). We investigated the production of NO, the gene expression of nitric oxide synthase (iNOS) and the morphology of isolated Kupffer cells (KC) and liver endothelial cells (EC) after treatment with killed Propionibacterium avidum (PPA), a Gram positive anaerobic bacterium. After isolation by centrifugal elutriation, KC and EC were cultivated for 24 h, then treated with different doses of PPA during 1-4 days. NO production was assessed using the Griess reagent. The expression of iNOS was detected by Northern blot with an iNOS-specific eDNA probe and the cellular morphology was investigated by transmission and scanning electron microscopy. We found that KC but not EC produce NO after treatment but interferon gamma pretreatment of the EC
35
of protection. Transforming growth factor-fl and epidermal growth factor, although induced MT did not seem to confer the same level of cytoprotection against cisplatin-induced toxicity in HPT cells. Work supported by the Wellcome Trust (Ref: 038369/Z/93/Z/ PMG/RB).
Keywords: metallothionein; proximal tubules; cisplatin; nephrotoxicity; human; in vitro
P1C-1301 MORPHOLOGICAL ALTERATIONS INDUCED BY IP1C-128
CADMIUM, MERCURY, ZINC AND BISMUTH ON CULTURED HUMAN PROXIMAL TUBULAR CELLS
1 VALUE OF LIVER TISSUE DOSlMETRY DATA TO
I
DESIGN AND INTERPRET IN VITRO (HEPATOCYTE) GAP JUNCTION STUDIES WITH PEROXISOMAL PROLIFERATING AGENTS
A.W. Lington 1, G.H. Kalimi 2, J.E. Klaunig 2, A.I. Nikiforov * 1
i Exxon Biomedical Sciences, Inc.; 2 Indiana University Peroxisomal proliferating agents (PPA), such as trichloroethylene (TCE) and di-2-ethylhexyl phthalate (DEHP), are non-genotoxic liver carcinogens in mice (DEHP, TCE) and/or rats (DEHP). In vitro studies employing hepatocyte cultures are being used for PPA with a focus on inhibition of gap junctional intercellular communication (GJIC). Inhibition of GJIC has been proposed as a promising assay for tumor promoters. DEHP is readily hydrolyzed to the monoester (MEHP) in vivo and MEHP is the main chemical found in rodent liver. DEHP (1000/zM) failed to inhibit GJIC inhibition in rat/mouse hepatocytes, most likely due to poor hydrolysis to MEHP, while MEHP (150-500 /zM) inhibited GJIC in rat/mouse and was readily metabolized in this system. TCE is extensively metabolized to trichloroacetic acid (TCA) in mice but not rats (in vivo, hepatocytes). TCE and TCA inhibited GJIC in mouse hepatocytes but not in rat hepatocytes. In conclusion, metabolic and tissue dosimetry data on PPA can assist in the design and data interpretation on whether GJIC effects in vitro are due to parent chemical or metabolites and also provide a sound basis for species differences.
I P 1I C - 1 2 9 I
INDUCTION OF METALLOTHIONEIN IN CULTURED HUMAN PROXIMAL TUBULAR CELLS CAN PROTECT AGAINST CISPLATIN INDUCED NEPHROTOXlCITY
Vicente RodiUa *, Jonathan P. Little, Naheed Afzal, Adrian T. Miles, Gabrielle M. Hawksworth. University of Aberdeen, Departments of
Medicine and Therapeutics and Biomedical Sciences, Foresterhill, Aberdeen AB9 2ZD, Scotland Metallothioneins (MTs) are low molecular weight metal binding proteins characterized by their high cysteine content. MTs are thought to play a role in the protection against heavy metal toxicity, may act as free radical scavengers and maybe be involved in the homeostasis of essential metals such as copper and zinc. These proteins are not only inducible by metals but also by exposure to a wide range of physical and chemical agents including stress, cytokines, growth factors, hormones and corticosteroids. Cisplatin is one of the most widely used anticancer agents whose dose limiting factor is its nephrotoxicity. Cisplatin produces DNA damage, generates free radicals and induces lipid peroxidation. In this study we investigated the possible effects of chemically-induced metallothionein as a prospective protective protein in cisplatin induced nephrotoxicity in man using cultured human proximal tubular (HPT) cells. The results of our studies demonstrate that dexamethasone, interleukin-6, prostaglandin Et, ZnCI2 and Bi(NO3)3 all induce metallothionein as demonstrated using immunocytochemistry. Maximal MT induction was achieved 48 hours after exposure to the chemical. The cytoprotection exerted by this induced metallothionein on cisplatin-treated HPT cultures was evaluated using the MTT assay and resulted in varying degrees
Vicente Rodilla *, Adrian T. Miles, Deborah Marshall ], Gabrielle M. Hawksworth. University of Aberdeen, Departments of Medicine
and Therapeutics and Biomedical Sciences, Foresterhill, Aberdeen AB9 2ZD, Scotland; I Electron Microscopy Unit, Department of Medical Microbiology, Foresterhill, Aberdeen AB9 2ZD, Scotland The kidney in particular the proximal convoluted tubule is a major target for the toxic effects of various metals. In the present study we have investigated the toxicity of several inorganic metal compounds (CdCI2, HgC12 ZnCI2 and Bi(NO3)3) upon cultured human proximal tubular (HPT) cells. These cells were isolated from the cortex of nephrectomy specimens and purified by centrifugation in Percoll. The toxic effects of various concentrations of the metals were determined using the MTT assay. Mercury was the most toxic compound, followed by cadmium, zinc, and bismuth, in that order. The morphological alterations caused by exposure of the cells to the metals were determined using optical and electron microscopy (SEM). Although exposure to the metals caused varying effects, the treated cells acquired a spherical morphology. Exposure to mercury caused other morphological alterations such as elongation and irregular distortion of the cells. Additional SEM observations revealed a number of alterations at membrane level such as blisters and other protuberances including small blebs. In the case of cells exposed to CdCI2 and ZnC12 the integrity of the cell membrane was destroyed and holes and small craters appeared, eventually leading to the destruction of the cell. This work has been supported by the Wellcome Trust (Refs: 038369/Z/93/Z/PMG/RB and 046936)
Keywords: heavy metal toxicity; proximal tubules; human; in vitro; SEM I
I P1 C-1 31 I I
NITRIC OXIDE SYNTHASE GENE INDUCTION BY KILLED PROPIONIBACTERIUM A VIDUM IN RAT KUPFFER AND ENDOTHELIAL CELLS IN VITRO
Mounir Dhouib * l, Cathy Royer 2, Jean-Louis Gendrault 2, Alain Lugnier i. i University Louis Pasteur, Faculty of Pharmacy,
Department of Toxicology and Faculty of Medicine, Strasbourg, France; 2 University Louis Pasteur, Faculty of Pharmacy, Laboratory of Virology and INSERM U74, Strasbourg, France Macrophages protect against bacterial infections by the production of reactive molecules such as nitric oxide (NO). We investigated the production of NO, the gene expression of nitric oxide synthase (iNOS) and the morphology of isolated Kupffer cells (KC) and liver endothelial cells (EC) after treatment with killed Propionibacterium avidum (PPA), a Gram positive anaerobic bacterium. After isolation by centrifugal elutriation, KC and EC were cultivated for 24 h, then treated with different doses of PPA during 1-4 days. NO production was assessed using the Griess reagent. The expression of iNOS was detected by Northern blot with an iNOS-specific eDNA probe and the cellular morphology was investigated by transmission and scanning electron microscopy. We found that KC but not EC produce NO after treatment but interferon gamma pretreatment of the EC