RESUMO
ROS, RNS, BRIs and ROS-RNS hybrids are produced during drug or chemical metabolism in vivo. These reactive species are instrumental to the culmination of cellular oxidative stress (OS). OS, once turned on, does not spare any vital intracellular macromolecule, such as glutathione, DNA, RNA, proteins, enzymes, lipids and ATP. Since concentration gradients of such components are very delicately balanced for normal cellular functioning, a gross perturbation leads to cell injury and cell death. Abundant evidence now suggests that intracellular antioxidants keep OS in check and maintain homeostasis. Our laboratory has focused on the role of OS in orchestrating various forms of cell death during drug and chemically-induced target organ toxicity and their counteraction by various natural or synthetic antioxidants in in vivo models. Despite complexity of the in vivo models, results show that metabolism of xenobiotics are invariably associated with different degrees of OS and natural antioxidants such as grape seed extract, bitter melon extract (Momordica charantia) and N-acetylcysteine (NAC) which were very effective in counteracting organ toxicities by minimizing events linked to OS (lipid peroxidation and total glutathione), and CAD-mediated DNA fragmentation. Phytoextract exposure rescued cells from toxic assaults, protected genomic integrity, and minimized apoptotic, necrotic and apocrotic (oncotic necrosis) cell deaths. Pre-exposure mode was more effective than post-exposure route. Overall scenario suggests that OS may have been the prime modulator of death and/or survival programs, whereas, antioxidants may have imparted a dual role in either erasing death signals or reviving survival signals, and a combination of antioxidants may be more beneficial than a single entity to influence a number of intracellular events operating simultaneously to neutralize chaotic toxicological consequences.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fígado/patologia , Estresse Oxidativo/fisiologia , Extratos Vegetais/farmacologia , Acetaminofen/administração & dosagem , Acetaminofen/toxicidade , Administração Oral , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Furosemida/administração & dosagem , Furosemida/toxicidade , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fitoterapia , Extratos Vegetais/administração & dosagem , Espécies Reativas de Oxigênio/metabolismoRESUMO
Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications. Previous studies in our laboratory have demonstrated enhanced production of reactive oxygen species, lipid peroxidation and DNA fragmentation in both in vitro and in vivo models following treatment with naphthalene. Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. In this study, we have investigated the ability of 1 mM melatonin to protect against naphthalene-induced oxidative stress and DNA damage in cultured macrophage J774A.1 cells. No significant changes were observed when these macrophage cells were treated with 100 microM naphthalene. Approximately 2.0-, 4.2- and 4.4-fold increases in cytochrome c reduction were observed at 200, 400 and 500 mM concentrations of naphthalene, demonstrating the increased production of superoxide anion. At 24 h, lipid peroxidation increased by approximately 1.4-, 2.1- and 2.2-fold following treatment of these cells with 200, 400 and 500 mM concentrations of naphthalene, respectively, while 1.6-, 2.8- and 2.8-fold increases in DNA fragmentation were observed at these same concentrations. Two hour pretreatment of these cultured cells with 1 mM melatonin provided approximately 26-44% decreases in lipid peroxidation, superoxide anion production and DNA fragmentation in cells treated with 400 and 500 microM naphthalene. Cellular viability decreased significantly when cells were incubated with concentrations of naphthalene greater than 100 microM, while preincubation with melatonin significantly increased the cellular viability. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of reactive oxygen free radicals, resulting in lipid peroxidation and DNA damage, while preincubation with melatonin significantly suppressed cytoxicity in J774A.1 macrophage cells.
Assuntos
Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Macrófagos/efeitos dos fármacos , Melatonina/farmacologia , Naftalenos/antagonistas & inibidores , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Acetaminophen (AAP), the analgesic hepatotoxicant, is a powerful inducer of oxidative stress, DNA fragmentation, and apoptosis. The anti-apoptotic oncogene bcl-XL, and the pro-apoptotic oncogene p53 are two key regulators of cell cycle progression and/or apoptosis subsequent to DNA damage in vitro and in vivo. This study investigated the effect of AAP on the expression of these oncogenes and whether agents that modulate DNA fragmentation (chlorpromazine, CPZ) and DNA repair through poly(ADP-Ribose) polymerase (PARP) activity (4-AB: 4-aminobenzamide) can protect against AAP-induced hepatotoxicity by inhibiting oxidative stress, DNA fragmentation, and/or by altering the expression of bcl-XL and p53. In addition, the protective effect of supplemental nicotinamide (NICO), known to be depleted in cells with high PARP activity during DNA repair, is similarly evaluated. Male ICR mice (3 months old) were administered vehicle alone; nontoxic doses of 4-AB (400 mg/kg, ip), NICO (250 mg/kg, ip) or CPZ (25 mg/kg, ip), hepatotoxic dose of AAP alone (500 mg/kg, ip), or AAP plus one of the protective agents 1 h later. All animals were sacrificed 24 h following AAP administration. Serum alanine aminotransferase activity (ALT), hepatic histopathology and lipid peroxidation, DNA damage, and expression of bcl-XL and p53 (western blot analysis) were compared in various groups. All of the three agents significantly prevented AAP-induced liver injury, lipid peroxidation, DNA damage, and associated apoptotic and necrotic cell deaths, 4-AB being the most effective and NICO the least. Compared to control, there was a considerable decrease in bcl-XL expression, and an increase in p53 expression in AAP-exposed livers. The effect of AAP on bcl-XL was antagonized and that on p53 was synergized by the PARP-modulator 4-AB as well as NICO, whereas the endonuclease inhibitor CPZ was without effect on either bcl-XL or p53 expression. These results suggest that the hepatotoxic effect of AAP involves multiple mechanisms including oxidative stress, upregulation of endonuclease (or caspase-activated DNAse) and alteration of pro- and anti-apoptotic oncogenes. The observed antagonism of AAP-induced hepatocellular apoptosis and/or necrosis by modulators of multiple processes including DNA repair suggests the likelihood that a more effective therapy against AAP intoxication should involve a combination of antidotes.
Assuntos
Ácido 4-Aminobenzoico/toxicidade , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Clorpromazina/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Niacinamida/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , para-Aminobenzoatos , Alanina Transaminase/sangue , Animais , Benzamidas , Biomarcadores/sangue , Western Blotting , Calmodulina/antagonistas & inibidores , Dano ao DNA , Fragmentação do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/patologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Radiossensibilizantes/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína bcl-XRESUMO
Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Rim/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Endodesoxirribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/sangue , Camundongos , Camundongos Endogâmicos ICR , Necrose , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/sangueRESUMO
Previous studies from our laboratories have linked the protective abilities of IH636 grape seed proanthocyanidin extract (GSPE) with inactivation of anti-apoptotic gene bcl-XL, and modification of several other critical molecular targets such as DNA-damage/DNA-repair, lipid peroxidation and intracellular Ca2+ homeostasis. Especially, GSPE provided dramatic protection against acetaminophen (APAP)-induced hepatotoxicity, significantly increased bcl-XL expression in the liver, and antagonized both necrotic and apoptotic deaths of liver cells in vivo. However, it was not clear from this study whether anti-apoptogenic and anti-necrotic effects of GSPE were: (i) due to its interference with endonuclease activity, (ii) due to its antioxidant effect, or, (iii) due to its ability to inhibit microsomal drug metabolizing enzyme(s), such as CYP-4502E1. Since CYP-4502E1 primarily metabolizes acetaminophen in mice and rats, this study specifically focused on CYP-4502E1's catalytic activity in vitro. Overall this investigation compared the in vitro aniline hydroxylation patterns of: (i) in vivo GSPE-exposed and unexposed (control) mouse liver microsomes, (ii) induced (1% acetone in drinking water for 3 days) and uninduced rat liver microsomes in the presence and absence of GSPE in vitro, and (iii) control rat liver microsomes in the presence of an anti-APAP agent 4-aminobenzamide (4-AB) in vitro. For the in vivo assessment, male B6C3F1 mice were fed GSPE diet (ADI 100 mg/kg body wt) for 4 weeks, and liver microsomes were isolated from both control and GSPE-fed mice for aniline hydroxylation, a specific marker of CYP-4502E1 activity. Data show that hydroxylation was 40% less in microsomes from GSPE-exposed livers compared to control microsomes. Similarly, when rat liver microsomes were incubated with various concentrations of GSPE in vitro (100 and 250 microg/ml), aniline hydroxylation was inhibited to various degrees (uninduced: 40 and 60% and induced: 25 and 50%, respectively with 100 and 250 microg/ml). Influence of GSPE on hydroxylation patterns were compared with another hepatoprotective agent 4-aminobenzamide (4-AB), a well-known modulator of nuclear enzyme poly(ADP-ribose) polymerase, and the data shows that 4-AB did not alter aniline hydroxylation at all. Collectively, these results may suggest that GSPE has the ability to inhibit CYP-4502E1, and this is an additional cytoprotective attribute, in conjunction with its novel antioxidant and/or antiendonucleolytic potential.
Assuntos
Ácido 4-Aminobenzoico/farmacologia , Acetaminofen/farmacologia , Compostos de Anilina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citoproteção/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Extratos Vegetais/farmacologia , para-Aminobenzoatos , Acetona/administração & dosagem , Acetona/farmacologia , Administração Oral , Animais , Benzamidas , Disponibilidade Biológica , Catálise/efeitos dos fármacos , Reparo do DNA , Relação Dose-Resposta a Droga , Extrato de Sementes de Uva , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Extratos Vegetais/administração & dosagem , Proantocianidinas , Ratos , Ratos Sprague-DawleyRESUMO
Grape seed proanthocyanidins have been demonstrated to exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. In our previous studies, IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) demonstrated excellent concentration- and dose-dependent free radical scavenging abilities in both in vitro and in vivo models and provided significantly better protection than vitamins C, E and beta-carotene. GSPE demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells and macrophage J774A.1 cells. In this study, the bioavailability and protective ability of GSPE were examined against acetaminophen-induced hepatoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, cadmium chloride-induced nephrotoxicity, dimethylnitrosamine-induced spleenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity in mice. In each experiment, half of the test animals were orally fed GSPE for 7-10 days prior to drug/chemical exposure, while the other half received no GSPE. Parameters of analysis included changes in serum chemistry [alanine amino-transferase (ALT), blood urea nitrogen and creatine kinase], histopathology and integrity of genomic DNA. The results indicated that GSPE preexposure prior to the drugs/chemicals such as acetaminophen, amiodarone, doxorubicin, cadmium chloride or dimethylnitrosamine treatment, provided near complete protection in terms of serum chemistry changes (ALT, blood urea nitrogen and creatine kinase) and inhibition of both forms of cell death, e.g., apoptosis and necrosis. DNA damage in various tissues triggered by these agents was significantly reduced. Histopathological examination of the organs evaluated reflected similar patterns to those of the serum chemistry and DNA results. MOCAP exposure showed symptoms of severe neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic change or brain pathology. GSPE afforded only partial protection in the brain tissue. These results suggest that GSPE exposure is bioavailable and provides significant multiorgan protection against drug- and chemical-induced toxic assaults.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Sequestradores de Radicais Livres/uso terapêutico , Extratos Vegetais/uso terapêutico , Alanina Transaminase/sangue , Animais , Antioxidantes/farmacocinética , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Disponibilidade Biológica , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Sequestradores de Radicais Livres/farmacocinética , Sequestradores de Radicais Livres/farmacologia , Extrato de Sementes de Uva , Humanos , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Hepatopatias/prevenção & controle , Pneumopatias/induzido quimicamente , Pneumopatias/prevenção & controle , Masculino , Camundongos , Extratos Vegetais/farmacocinética , Extratos Vegetais/farmacologia , Proantocianidinas , Baço/lesões , Baço/patologiaRESUMO
Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.
Assuntos
Antocianinas/farmacologia , Antioxidantes/farmacologia , Radicais Livres/antagonistas & inibidores , Extratos Vegetais/farmacologia , Proantocianidinas , Animais , Antocianinas/farmacocinética , Antioxidantes/farmacocinética , Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Disponibilidade Biológica , Doenças Cardiovasculares/prevenção & controle , Relação Dose-Resposta a Droga , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacocinética , Sequestradores de Radicais Livres/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Nefropatias/prevenção & controle , Hepatopatias/prevenção & controle , Neoplasias/prevenção & controle , Extratos Vegetais/química , Sementes/química , Vitamina E/farmacologia , beta Caroteno/farmacologiaRESUMO
The protein BCL-X(L) and protein product of proto-oncogene bcl-2 act as apoptosis antagonists, and BCL-X(S) serve as a dominant death promoter, including apoptosis following exposure to chemotherapeutic drugs. This investigation examined whether some aspects of the highly integrated process of acetaminophen (AAP)-induced hepatotoxicity involve down-regulation or upregulation of expression of BCL-2, BCL-X(L) and BCL-X(S) in mouse liver in vivo. Male ICR mice (CD-1; 35-45 g) were treated ip with a hepatotoxic dose of AAP (500 mg/kg) and sacrificed 0, 6, and 18 h later. Blood was collected upon sacrifice for determination of serum alanine aminotransferase (ALT) activity and the liver was sectioned for histopathological diagnosis of necrosis/apoptosis. Portions of liver tissues were also used for DNA extraction (for gel electrophoresis) and Western blot analysis. This study demonstrates that administration of a hepatotoxic dose of AAP to ICR mice results in severe liver injury (ALT leakage >200-fold at 6 h and >600-fold at 18 h) leading to massive cell death by apoptosis (diagnosed by nuclear ultrastructure, histopathology, and DNA ladder), in addition to necrosis coupled with spectacular changes in the BCL-X(L) expression (6 and 18 h after AAP administration). Western blot analysis of the liver proteins revealed that mouse liver expresses two proteins, BCL-X(L) and BCL-X(S), and does not express BCL-2. As the toxicity progressed, during 6 and 18 h post-AAP administration, the BCL-X(L) protein band shifted to a slower mobility band which might represent a phosphorylated form of BCL-X(L). Appearance of this higher molecular weight BCL-X(L) protein band correlated with massive apoptotic death of liver cells along with ladder-like DNA fragmentation. In the same time period, death inhibitory gene bcl-2 remained unexpressed, and the level of expression of BCL-X(S) remained unaltered. Whether the consistent level of expression of BCL-X(S) reflected inability of AAP to influence its expression remains unknown. Unaltered expression of BCL-X(S) in the near total absence of BCL-2 expression raises questions regarding the death promoting role of BCL-X(S) in vivo. The precise role of modified form of BCL-X(L) remains elusive. However, this study may have demonstrated for the first time drug-induced changes in the expression of anti-apoptotic gene BCL-X(L), and a positive link between AAP-induced apoptotic death and modification of BCL-X(L) protein in vivo.
Assuntos
Acetaminofen/toxicidade , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Alanina Transaminase/sangue , Animais , Apoptose/genética , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , DNA/análise , Fragmentação do DNA , Regulação da Expressão Gênica , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Necrose , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-XRESUMO
Several observations, both in humans and laboratory animals, have suggested that proanthocyanidins exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. Specifically, some of our earlier studies have shown that IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) provides excellent concentration- and dose-dependent protection against toxicities induced by diverse agents, such as acetaminophen, hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate (TPA), smokeless-tobacco extract, idarubicin and 4-hydroxyperoxycyclophosphamide in both in vitro and in vivo models. In some models, GSPE proved to be a better cytoprotectant than vitamins C, E and beta-carotene. The purpose of this investigation was three fold: (i) to indirectly assess the bioavailability of GSPE in multiple target organs, (ii) quantify GSPE's capacity to avert cadmium chloride (CdCl2)-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity, and lastly (iii) to evaluate possible mechanisms of protection in mice. In order to determine all these, three separate experiments were designed and each experiment consisted of four groups, such as vehicle control, GSPE alone, toxicant alone and GSPE + toxicant. GSPE was administered orally (100 mg/Kg) for 7-8 days prior to the toxicant exposure. Parameters of the analyses included evaluation of serum chemistry changes (ALT, BUN and CK), histopathology and integrity of genomic DNA, both quantitatively and qualitatively. Results indicate that GSPE preexposure prior to cadmium chloride and DMN provided near complete protection in terms of serum chemistry changes (ALT, BUN and CK) and inhibition of both forms of cell death. e.g., apoptosis and necrosis. DNA damage, a common denominator usually associated with both apoptosis and necrosis was significantly reduced by GSPE treatment. Histopathological examination of organs correlated strongly with the changes in serum chemistry and the DNA modification data. Surprisingly, MOCAP exposure showed symptoms of neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic DNA damage or brain pathology. Although, GSPE appeared to partially protect the neural tissue, it powerfully antagonized MOCAP-induced mortality. Taken together, this study suggests that in vivo GSPE-preexposure may protect multiple target organs from a variety of toxic assaults induced by diverse chemical entities.
Assuntos
Ativinas/farmacologia , Ativinas/uso terapêutico , Cloreto de Cádmio/toxicidade , Dimetilnitrosamina/toxicidade , Nefropatias/prevenção & controle , Doenças do Sistema Nervoso/induzido quimicamente , Organotiofosfatos/toxicidade , Extratos Vegetais/farmacologia , Baço/efeitos dos fármacos , Ativinas/administração & dosagem , Administração Oral , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Cloreto de Cádmio/administração & dosagem , Creatina Quinase/sangue , Fragmentação do DNA/efeitos dos fármacos , Dimetilnitrosamina/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Extrato de Sementes de Uva , Injeções Intraperitoneais , Nefropatias/induzido quimicamente , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Necrose , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/prevenção & controle , Organotiofosfatos/administração & dosagem , Proantocianidinas , Baço/patologiaRESUMO
Grape seed extract, primarily a mixture of proanthocyanidins, has been shown to modulate a wide-range of biological, pharmacological and toxicological effects which are mainly cytoprotective. This study assessed the ability of IH636 grape seed proanthocyanidin extract (GSPE) to prevent acetaminophen (AAP)-induced nephrotoxicity, amiodarone (AMI)-induced lung toxicity, and doxorubicin (DOX)-induced cardiotoxicity in mice. Experimental design consisted of four groups: control (vehicle alone), GSPE alone, drug alone and GSPE+drug. For the cytoprotection study, animals were orally gavaged 100 mg/Kg GSPE for 7-10 days followed by i.p. injections of organ specific three drugs (AAP: 500 mg/Kg for 24 h; AMI: 50 mg/Kg/day for four days; DOX: 20 mg/Kg for 48 h). Parameters of study included analysis of serum chemistry (ALT, BUN and CPK), and orderly fragmentation of genomic DNA (both endonuclease-dependent and independent) in addition to microscopic evaluation of damage and/or protection in corresponding PAS stained tissues. Results indicate that GSPE preexposure prior to AAP, AMI and DOX, provided near complete protection in terms of serum chemistry changes (ALT, BUN and CPK), and significantly reduced DNA fragmentation. Histopathological examination of kidney, heart and lung sections revealed moderate to massive tissue damage with a variety of morphological aberrations by all the three drugs in the absence of GSPE preexposure than in its presence. GSPE+drug exposed tissues exhibited minor residual damage or near total recovery. Additionally, histopathological alterations mirrored both serum chemistry changes and the pattern of DNA fragmentation. Interestingly, all the drugs, such as, AAP, AMI and DOX induced apoptotic death in addition to necrosis in the respective organs which was very effectively blocked by GSPE. Since AAP, AMI and DOX undergo biotransformation and are known to produce damaging radicals in vivo, the protection by GSPE may be linked to both inhibition of metabolism and/or detoxification of cytotoxic radicals. In addition, its' presumed contribution to DNA repair may be another important attribute, which played a role in the chemoprevention process. Additionally, this may have been the first report on AMI-induced apoptotic death in the lung tissue. Taken together, these events undoubtedly establish GSPE's abundant bioavailability, and the power to defend multiple target organs from toxic assaults induced by structurally diverse and functionally different entities in vivo.
Assuntos
Acetaminofen/toxicidade , Amiodarona/toxicidade , Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , Cardiopatias/prevenção & controle , Nefropatias/prevenção & controle , Pneumopatias/prevenção & controle , Extratos Vegetais/farmacologia , Rosales , Acetaminofen/administração & dosagem , Administração Oral , Alanina Transaminase/sangue , Amiodarona/administração & dosagem , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Nitrogênio da Ureia Sanguínea , Creatina Quinase/sangue , Fragmentação do DNA/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Sequestradores de Radicais Livres , Extrato de Sementes de Uva , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Injeções Intraperitoneais , Nefropatias/induzido quimicamente , Nefropatias/patologia , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Masculino , Camundongos , Necrose , ProantocianidinasRESUMO
Several molecular events in the apoptotic or necrotic death of hepatocytes induced by acetaminophen (AAP) now appear to be well defined. Recent studies also indicate that select expression of bcl-Xl is possibly modified during AAP-induced liver injury. The purpose of this study was several-fold: (i) to examine the hepatoprotective ability of short-term (3-day) and long-term (7-day) exposures of a grape seed proanthocyanidin extract (GSPE) on AAP-induced liver injury and animal lethality; (ii) to monitor effects of GSPE on one of the prime targets of AAP, i.e., hepatocellular genomic DNA and associated apoptotic and necrotic death; and (iii) to unravel changes in the pattern of expression of an antiapoptotic gene, bcl-Xl in the liver. In order to investigate these events, male ICR mice (30-40 g) were administered nontoxic doses of GSPE (3 or 7 days, 100 mg/kg, po), followed by hepatotoxic doses of AAP (400 and 500 mg/kg, ip), and sacrificed 24 h later. Serum was analyzed for alanine aminotransferase activity (ALT) and the liver for histopathological diagnosis of apoptosis/necrosis. The ability of AAP to promote apoptotic DNA fragmentation and its counteraction by GSPE in the liver was also evaluated quantitatively (by a sedimentation assay) and qualitatively (by agarose gel electrophoresis). Portions of livers were also subjected to Western blot analysis (27,000g fraction of liver homogenates) to examine the pattern of expression of cell death inhibitory gene bcl-Xl. Results indicate that 7-day GSPE preexposure induced dramatic protection and markedly decreased liver injury and animal lethality culminated by AAP, when compared to a short-term 3-day exposure. Abrogation of toxicity was also mirrored in DNA fragmentation. Histopathological evaluation of liver sections showed remarkable counteraction of AAP-toxicity by this novel GSPE and substantial inhibition of both apoptotic and necrotic liver cell death. Agarose gel electrophoresis revealed that 7-day GSPE preexposure prior to AAP administration completely blocked Ca(2+)/Mg(2+)-Ca(2+)/Mg(2+)-dependent-endonuclease-mediated ladder-like fragmentation of genomic DNA and significantly altered the bcl-Xl expression. The most dramatic changes observed in this study were: (i) substantial increase in the expression of bcl-Xl in the liver by 7-day GSPE exposure alone; (ii) significant modification bcl-Xl expression by AAP alone; and (iii) dramatic inhibition of AAP-induced modification of bcl-Xl (phosphorylation?) expression by GSPE. In summary, these observations demonstrate that GSPE preexposure may significantly attenuate AAP-induced hepatic DNA damage, apoptotic and necrotic cell death of liver cells, and, most remarkably, antagonize the influence of AAP-induced changes in bcl-Xl expression in vivo.
Assuntos
Acetaminofen/farmacologia , Antocianinas/farmacologia , Apoptose/fisiologia , Morte Celular/fisiologia , Fígado/citologia , Proantocianidinas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Rosales , Extratos de Tecidos/farmacologia , Alanina Transaminase/sangue , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Sementes , Proteína bcl-XRESUMO
We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.
Assuntos
Antioxidantes/farmacologia , Apoptose , Queratinócitos/citologia , Queratinócitos/fisiologia , Peroxidação de Lipídeos , Mucosa Bucal/citologia , Mucosa Bucal/fisiologia , Estresse Oxidativo , Plantas Tóxicas , Proantocianidinas , Tabaco sem Fumaça/farmacologia , Antocianinas/farmacologia , Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Células Cultivadas , Fragmentação do DNA , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , NADH Desidrogenase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Rosales , Sementes , Superóxidos/metabolismo , Vitamina E/farmacologiaRESUMO
The study was designed to investigate the feasibility of developing a transdermal drug dosage form of promethazine hydrochloride (PMH). The in vitro release and diffusion characteristics of PMH from various dermatological polymeric bases were studied using cellulose membrane and hairless mouse skin as the diffusion barriers. These included polyethylene glycol (PEG), hydroxypropyl methylcellulose (HPMC), cross-linked microcrystalline cellulose, and carboxyl methyl cellulose sodium (Avicel CL-611), and a modified hydrophilic ointment USP. In addition, the effects of several additive ingredients known to enhance the drug release from topical formulations were evaluated. The general rank order for the drug release from these formulations using cellulose membrane was observed to be PEG > HMPC > Avicel CL-611 > hydrophilic ointment base. The inclusion of the additives had little or no effect on the drug diffusion from these bases, except for the hydrophilic ointment formulation containing 15% ethanol, which provided a significant increase in the drug release. However, when these formulations were studied for drug diffusion through the hairless mouse skin, the Avicel CL-611 base containing 15% ethanol exhibited the optimum drug release. The data also revealed that this formulation gave the highest steady-state flux, diffusion, and permeability coefficient values and correlated well with the amount of drug release.
Assuntos
Química Farmacêutica , Antagonistas dos Receptores Histamínicos H1/administração & dosagem , Polímeros , Prometazina/administração & dosagem , Administração Cutânea , Animais , Permeabilidade da Membrana Celular , Celulose , Composição de Medicamentos , Estudos de Viabilidade , Antagonistas dos Receptores Histamínicos H1/farmacocinética , Camundongos , Camundongos Pelados , Pomadas , Prometazina/farmacocinética , Pele/metabolismoRESUMO
1. The comparative protective abilities of zinc L-methionine, zinc DL-methionine, zinc sulfate, zinc gluconate, L-methionine, DL-methionine, and vitamin E succinate (VES) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation, DNA fragmentation, and glutathione depletion in the hepatic and brain tissues, and production of reactive oxygen species by peritoneal macrophages were assessed. In addition, mice were fed a zinc-deficient diet for 5 weeks, and treated with TPA and/or zinc L-methionine or zinc DL-methionine, and similar studies were conducted. 2. The zinc-deficient diet induced oxidative stress in the hepatic and brain tissues as well as in the peritoneal macrophages as evidenced by significantly enhanced lipid peroxidation. DNA fragmentation, glutathione depletion, and production of reactive oxygen species. 3. Treatment of mice with zinc L-methionine, zinc DL-methionine, and VES decreased TPA-induced reactive oxygen species production as evidenced by significant decreases in chemiluminescence in peritoneal macrophages by approximately 45%, 31%, and 47%, respectively, and cytochrome c reduction by approximately 54%, 35%, and 41%, respectively, as compared with control values. Similar results were observed with liver and brain lipid peroxidation, DNA fragmentation, and glutathione depletion. 4. Zinc salts and antioxidants provided significant protection against TPA-induced oxidative damage. Zinc L-methionine provided the best protection.
Assuntos
Encéfalo/efeitos dos fármacos , Dano ao DNA , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/toxicidade , Compostos de Zinco/farmacologia , Animais , Encéfalo/metabolismo , Grupo dos Citocromos c/metabolismo , Interações Medicamentosas , Feminino , Gluconatos/farmacologia , Fígado/metabolismo , Medições Luminescentes , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Oxirredução , Tocoferóis , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Sulfato de Zinco/farmacologiaRESUMO
Oxygen free radicals (OFR) are implicated in the pathogenesis of stress, chemically induced gastric lesions, and gastrointestinal injury. The concentration-dependent scavenging abilities of bismuth subsalicylate (SBS), colloidal bismuth subcitrate (CBS), and selected OFR scavengers, including superoxide dismutase (SOD), catalase, mannitol, and allopurinol were examined against biochemically or chemically generated superoxide anion, hydroxyl radical, and hypochlorite radical plus hypochlorous acid based on a chemiluminescence assay. Furthermore, both gastric (GM) and intestinal mucosa (IM) were individually exposed in vitro to these free radical generating systems, and the concentration-dependent protective abilities of SBS and CBS against lipid peroxidation (LP) were compared with selected OFR scavengers. In addition, 24-hr fasted rats were orally treated with the necrotizing agents 0.6 M HCl, 0.2 M NaOH, 80% ethanol, and aspirin (200 mg/kg). The extent of tissue injury in the GM and IM was determined by assessing LP, DNA fragmentation, and membrane microviscosity. Dose- and time-dependent in vivo protective abilities of CBS (100 mg/kg) and SBS (15 mg/kg) were also assessed. Following incubations with superoxide anion and hydroxyl radical generating systems in the presence of 125 mg SBS/liter, approximately 47% and 61% inhibitions were observed in the chemiluminescence response, respectively, while 48% and 46% inhibitions were observed with 125 mg CBS/liter. SBS and CBS exerted similar abilities towards hypochlorite radical plus hypochlorous acid. Approx. 3.1- and 3.7-fold increases in LP were observed in the GM and IM of rats following oral administration of 0.6 M HCl. Pretreatment of the rats with SBS and CBS decreased 0.6 M HCl-induced LP in the GM by approx. 39% and 27%, respectively, with similar decreases in LP in the IM. SBS exhibited better protective abilities towards 0.6 M HCl and 0.2 m NaOH-induced GM and IM injury as compared to CBS. SBS and CBS provided similar protection towards 80% ethanol-induced gastric injury, while CBS exerted a superior protective ability towards aspirin-induced gastric injury. The results demonstrate that both SBS and CBS can scavenge reactive oxygen species and prevent tissue damage produced by OFR.
Assuntos
Bismuto/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Espécies Reativas de Oxigênio , Animais , Antiulcerosos/farmacologia , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Medições Luminescentes , Compostos Organometálicos/farmacologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Salicilatos/farmacologiaRESUMO
In addition to its use as a stabilizer/rigidifier of membranes, cholesteryl hemisuccinate, tris salt (CS) administration has also been shown to protect rats from the hepatotoxic effects of carbon tetrachloride (CCl4). To further our understanding of the mechanism of CS cytoprotection, we examined in rats and mice the protective abilities of CS and the non-hydrolyzable ether form of CS, gamma-cholesteryloxybutyric acid, tris salt (CSE) against acetaminophen-, adriamycin-, carbon tetrachloride-, chloroform- and galactosamine-induced toxicity. The results of these studies demonstrated that CS-mediated protection is not selective for a particular species, organ system or toxic chemical. A 24-h pretreatment of both rats and mice with a single dose of CS (100mg/kg, i.p.), resulted in significant protection against the hepatotoxic effects of CCl4, CHCl3, acetaminophen and galactosamine and against the lethal (and presumably cardiotoxic) effect of adriamycin administration. Maximal CS-mediated protection was observed in experimental animals pretreated 24 h prior to the toxic insult. These data suggest that CS intervenes in a critical cellular event that is an important common pathway to toxic cell death. The mechanism of CS protection does not appear to be dependent on the inhibition of chemical bioactivation to a toxic reactive intermediate (in light of the protection observed against galactosamine hepatotoxicity). However, based on the data presented, we can not exclude the possibility that CS administration inhibits chemical bioactivation. Our findings do suggest that CS-mediated protection is dependent on the action of the intact anionic CS molecule (non-hydrolyzable CSE was as protective as CS), whose mechanism has yet to be defined.
Assuntos
Acetaminofen/toxicidade , Tetracloreto de Carbono/toxicidade , Clorofórmio/toxicidade , Ésteres do Colesterol/uso terapêutico , Doxorrubicina/toxicidade , Galactosamina/toxicidade , Hepatopatias/prevenção & controle , Animais , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Cardiopatias/prevenção & controle , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-DawleyRESUMO
This study of acetaminophen (AAP) hepatotoxicity examined whether some aspects of the highly integrated process of drug-induced toxicity involves apoptosis, in addition to necrosis in vivo; and if so, whether cholesteryl hemisuccinate (CS) pretreatment would selectively interfere with apoptotic or necrotic liver cell death. We have previously demonstrated that CS preexposure in vivo, protects hepatocellular necrosis and necrosis-related events induced by carbon tetrachloride (CCl4) administration. Our study demonstrates that administration of hepatotoxic doses of AAP (350-500 mg/kg, i.p.) to ICR mice (CD-1) results in severe liver injury leading to cell death both by necrosis and apoptosis. AAP-induced cell death was preceded by massive elevation in serum alanine aminotransferase coupled with rapid loss of large genomic DNA (2-24 hr), fragmentation of DNA in the form of a ladder (2-24 hr), apoptotic nuclear condensation at early hours (2-6 hr) followed by massive fragmentation and margination of heterochromatin at later (6-24) hours and a near total loss of glycogen in pericentral areas. Although CS (100 mg/kg, i.p.) alone had no noticeable biochemical or morphological effects, its administration before AAP (350-500 mg/kg, i.p.) abrogated histological and biochemical diagnostics of both apoptosis and necrosis. These include near total absence of loss of large genomic DNA and glycogen, and dramatic protection from escalating levels of liver injury. CS pretreatment also arrested AAP-induced ultrastructural changes typical of both apoptosis and necrosis. Histopathological examination of periodic acid-Schiff stained liver sections mirrored the biochemical and ultrastructural findings. In conclusion, this study for the first time establishes that apoptosis, in addition to necrosis, significantly contributes to AAP hepatotoxicity in vivo, and preexposure of mice to CS prevents AAP-induced hepatic apoptosis and necrosis.
Assuntos
Acetaminofen/farmacologia , Apoptose/efeitos dos fármacos , Ésteres do Colesterol/farmacologia , Fígado/efeitos dos fármacos , Acetaminofen/antagonistas & inibidores , Animais , Fragmentação do DNA , Fígado/patologia , Fígado/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica , NecroseRESUMO
Tacrine (tetrahydroaminoacridine) is currently the only drug approved for the treatment of Alzheimer's disease. Unfortunately, tetrahydroaminoacridine therapy is often limited by this drug's propensity to induce reversible hepatotoxicity. Using suspensions of freshly isolated rat hepatocytes, we investigated the mechanism of tetrahydroaminoacridine cytotoxicity by examining the effect of tetrahydroaminoacridine on hepatocyte viability, protein synthesis, protein, DNA and RNA levels and ultrastructure. Our experimental findings support the explanation that tetrahydroaminoacridine-induced hepatotoxicity results from tetrahydroaminoacridine's adverse effect on protein synthesis and ribosomal structure and function. We found that viable, tetrahydroaminoacridine-treated hepatocytes (1.0 to 2.0 mmol/L or 118 to 235 micrograms/10(6) cells) demonstrated a dose-dependent and dramatic aggregation of ribosomes on endoplasmic reticulum as well as the aggregation of other nucleic acids found in the nucleus (chromatin) and in mitochondria. These electron microscopy data suggest that tetrahydroaminoacridine treatment results in severe ribosomal dysfunction. This was confirmed by the observed rapid loss of cellular RNA content (but not DNA or protein) and the rapid and complete inhibition of protein synthesis in tetrahydroaminoacridine-treated cells (lowest concentration tested was 0.5 mmol/L or 58 micrograms/10(6) cells). Thus tetrahydroaminoacridine treatment appears to aggregate hepatocellular nucleic acids, and in doing so adversely affects ribosomal function and protein synthesis. We propose that these adverse effects of exposure to tetrahydroaminoacridine are responsible for tetrahydroaminoacridine-induced hepatotoxicity.
Assuntos
Fígado/efeitos dos fármacos , Biossíntese de Proteínas , Tacrina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Depressão Química , Fígado/citologia , Fígado/metabolismo , Masculino , RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestrutura , SuspensõesRESUMO
Previous studies from our laboratory have demonstrated that the administration of alpha-tocopheryl hemisuccinate (TS), but not unesterified alpha-tocopherol (T), protects hepatocytes from a variety of toxic insults including chemicals, drugs, metals, and oxidative stress. One possible mechanism for this unique cytoprotection is that succinate released from cellular TS is used as a supplemental energy source during a toxic challenge. To test this hypothesis, we examined the effect of TS (25 microM) administration on cell viability, lipid peroxidation, and several cellular energy-related processes such as mitochondrial membrane potential (MMP, psi delta), lactate formation, and ATP and K+ concentrations in isolated hepatocyte suspensions during a toxic challenge with the alkylating agent, ethyl methanesulfonate (EMS). Data from these studies demonstrate that EMS treatment results in rapid cell death and lipid peroxidation following 2 h of incubation. Preceding EMS-induced cell death was a rapid loss of MMP, intracellular ATP and K+ levels, and mitochondrial ultrastructure as well as a transient increase in cellular lactate production. Pretreatment of hepatocytes with TS prior to EMS exposure prevented the loss of MMP and mitochondrial ultrastructural changes as well as lipid peroxidation and cell death. Cellular ATP levels and lactate production did not reflect the protection afforded to TS-treated hepatocytes. Protection against EMS-induced toxicity was not observed when hepatocytes were: (i) pretreated with TS and esterase inhibitors (preventing T and succinate release from TS); (ii) pretreated with other lipophilic succinate derivatives (cholesteryl hemisuccinate, monomethyl and dimethyl succinate); or (iii) pretreated with T and sodium succinate. Unlike monomethyl succinate, cytoprotective TS pretreatment did not stimulate gluconeogenesis or glycolysis. Hepatocytes isolated from rats pretreated for 24 h with T were not protected from the toxic effects of EMS, unlike TS-pretreated rats. In conclusion, TS cytoprotection against the mitochondrial toxicant EMS appears to be related to the hepatocellular accumulation of TS and the maintenance of mitochondrial function (MMP). Based on our earlier findings and the present observations, we propose that a unique subcellular disposition for TS and the subsequent release of T and succinate at a critical mitochondrial site is responsible for the observed cytoprotection.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Metanossulfonato de Etila/farmacologia , Fígado/efeitos dos fármacos , Vitamina E/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Succinatos/metabolismo , Succinatos/farmacologia , Ácido Succínico , Tocoferóis , Vitamina E/farmacologiaRESUMO
The mechanism by which 7,12-dimethylbenz[a]anthracene (DMBA) produces cytotoxicity in lymphocytes was investigated in these studies using the murine A20.1 B cell lymphoma. Results show that in vitro exposure of these cells to 10-30 microM DMBA for 4 hr produced an increase in intracellular Ca2+, DNA fragmentation, and subsequent cell death. Elevation of Ca2+ and DNA fragmentation induced by DMBA were greatly pronounced when the A20.1 cells were exposed at high cell density (10(7) cells/ml). DMBA-induced DNA fragmentation and cell death were inhibited by coexposure of A20.1 cells to a calcium chelator (EDTA), a general nuclease and polymerase inhibitor (aurintricarboxylic acid), and a protein synthesis inhibitor (cycloheximide). These agents have been previously shown to inhibit apoptosis in lymphocytes and other cells exposed to chemical agents. We also found that cyclosporin A, an inhibitor of Ca(2+)-dependent pathways of T and B cell activation, prevented apoptosis in the A20.1 cell line. These results demonstrate that DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma by Ca(2+)-dependent pathways. The increased sensitivity of A20.1 at high cell density to Ca2+ elevation and DNA fragmentation suggests that cell to cell interactions may also be important in this process.