RESUMEN
The 2,4,5-triaryl imidazole derivatives (API) were designed, screened and characterized kinetically & thermodynamically against Pepsin and their activity was also tested on the in silico platform. The docking studies of API with Pepsin show that these are novel and unique inhibitors of Aspartic protease. Drug like properties of these compounds were validated in silico based on Lipinski's rule of Five by calculating ClogP, LogS, H-bond acceptors, H-Bond donors, rotational bonds, PSA, PB and BBB values. The Et/Ki and Et/Km values of API show that they follow the Michaelis-Menten kinetics. The binding of inhibitors with proteases was explained by using Van't Hoff plot and thermodynamic parameters viz. free energy (ΔG), Entropy (ΔS) and Enthalpy (ΔH). The Van't Hoff analysis showed that the value of Ki decreases with increase in temperature and the binding of the inhibitor are entropically driven. API act as new potent aspartic protease inhibitors with Ki, for Pepsin, ranges from 3.7 µM to 16.7 µM. Strong hydrophobic groups at C-4 & C-5 position in API favor binding of inhibitors with Pepsin. Experiments also showed that among C-2 aryl substituted imidazole, a 4-substitution on aryl ring is preferred and less polar substituent makes the molecule more active whereas polar substituents at 2-position on C-2 aryl ring makes the molecule less active. The docking studies of API with Pepsin further intensify and validate our results.
Asunto(s)
Proteasas de Ácido Aspártico/antagonistas & inhibidores , Diseño de Fármacos , Imidazoles/síntesis química , Imidazoles/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Proteasas de Ácido Aspártico/metabolismo , Relación Dosis-Respuesta a Droga , Imidazoles/química , Cinética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
BACKGROUND: Tocotrienol (Tocomin) are naturally occurring analogues of vitamin E family and has been reported to possess a potent free radical scavenging activity. In the present study we have initially investigated protective role of tocotrienol against infection and inflammation induced alterations in tissues antioxidant defense system, as well as speculated, via in silico docking studies, that tocotrienol can act by directly binding to antioxidant enzymes. MATERIALS AND METHODS: Syrian hamsters were injected with bacterial lipopolysaccharide (LPS, 200 microg), zymosan (20 mg), or turpentine (0.5 ml) to mimic acute infection, acute systemic inflammation, and acute localized inflammation, respectively, which are responsible for the generation of plenty of free radicals that causes oxidative stress. Tocomin (10 mg) was administered daily for 10 days before and 12 h after lipopolysaccharides (LPS) or 24 h after turpentine or zymosan injection. Molecular docking studies were performed using Autodock 4.0. RESULTS: Our results show a significant decrease in the activities of antiperoxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), as well as reduced glutathione (GSH), in liver and kidney of LPS, turpentine or zymosan stressed hamsters. Feeding of 10 mg Tocomin to stressed hamsters was quite effective in reversing/normalizing the altered levels of enzymatic and nonenzymatic antioxidants in liver and kidney. In order to explore the interaction between tocotrienol and antioxidant enzymes a molecular docking study was performed. The results showed good interaction in term of binding energy and inhibition constant in the following order GR > CAT > SOD > GST > GPx. CONCLUSION: Our in vivo and in silico results for the first time indicate that tocotrienol significantly alleviate the condition of oxidative stress not only by its potent free radical scavenging properties but also may be by interacting directly and strongly with antioxidant enzymes as proved by molecular docking simulations.
Asunto(s)
Antioxidantes/farmacología , Simulación por Computador , Inflamación/tratamiento farmacológico , Modelos Biológicos , Oxidorreductasas/antagonistas & inhibidores , Tocotrienoles/farmacología , Animales , Cricetinae , Modelos Animales de Enfermedad , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Eritrocitos/metabolismo , Glutatión/antagonistas & inhibidores , Inflamación/inducido químicamente , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacosRESUMEN
Cigarette smoke contains several oxidants and free radicals. In the present study, we examined the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in the lungs of female Sprague-Dawley rats exposed to side-stream cigarette smoke for 6 h a day, 7 days a week for 1, 2, 4 and 12 weeks in a whole body-exposure system. The samples were analyzed for 8-oxodG by 32P-postlabeling-TLC enrichment and HPLC-ECD techniques to confirm and compare results. Animals were sacrificed 15 h after the cessation of smoke exposure and lung DNA was isolated by phenol/Sevag extractions in the presence of the free radical traps, 8-hydroxyquinoline (6.8 mM) and N-t-butyl-alpha-phenyl nitrone (500 microM) to minimize artifactual formation of 8-oxodG during sample work up. Analysis of lung DNA by 32P-postlabeling-TLC showed 8-oxodG levels (mean +/- SE) of 1.45+/-0.24, 2.68+/-0.65, 2.23+/-0.28 and 2.93+/-0.54 per 106 nucleotides after 1, 2, 4 and 12 weeks of smoke exposure. The respective values in sham-treated rats were 2.76+/-0.19, 3.69+/-0.20, 1.44+/-0.43 and 2.84+/-0.45 per 106 nucleotides, suggesting no significant effect of smoke exposure on tissue levels of 8-oxodG. HPLC-ECD procedure yielded slightly higher values for 8-oxodG in all groups, however, again significant differences between sham and smoke-exposed groups were not detected. It is concluded that the chronic exposure to side-stream cigarette smoke does not enhance the formation of 8-oxodG in rat lungs.
Asunto(s)
Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Pulmón/efectos de los fármacos , Fumar/efectos adversos , 8-Hidroxi-2'-Desoxicoguanosina , Administración por Inhalación , Animales , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , ADN/metabolismo , Femenino , Pulmón/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
To use oxidative DNA lesions as biomarkers of exposure and disease in the human scenario, the assay should not only be sensitive but also applicable to small quantities of DNA that is obtained from human tissue biopsies. Previously, we reported a nonenrichment (32)P-postlabeling method for measuring the oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) [Devanaboyina, U., and Gupta, R. C. (1996) Carcinogenesis 17, 917-924]. Here we report a substantial improvement of the assay by enriching 8-oxo-dGp by TLC prior to (32)P-labeling. Thus, unmodified nucleotides were removed by 1-directional polyethyleneimine (PEI)-cellulose TLC in 0.2 M formic acid. 8-Oxo-dGp, retained close to the origin, was eluted in 2 M triethylammonium acetate (pH 7.0), lyophilized, (32)P-labeled, resolved by TLC, and quantitated. The 8-oxo-dG signal was found to increase linearly with increasing amount of DNA. Its recovery was found to be 55-70%, as determined by using synthetic 8-oxo-dGp. Since the modified assay allowed using larger quantities of DNA and higher specific activity [gamma-(32)P]ATP than those used in the nonenrichment procedure, the signal-to-noise ratio increased to 30-100:1 from 3-5:1. The assay has a sensitivity of detection of <1 8-oxo-dG/10(7) nucleotides using submicrogram to microgram DNA. The 8-oxo-dG levels (mean +/- SE; n = 5) in the liver, lung, heart, bladder, and trachea DNA of 3 month-old female Sprague-Dawley rats were found to be 1.05 +/- 0.24, 0.97 +/- 0.09, 0.75 +/- 0.15, 0.79 +/- 0.15, and 1.17 +/- 0.28 per 10(6) nucleotides, respectively.
Asunto(s)
Biomarcadores/análisis , Daño del ADN , Radioisótopos de Fósforo , Animales , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Distribución TisularRESUMEN
Dithiolethiones are currently one of the most promising classes of cancer chemopreventive agents that exhibit antitumorigenic properties at numerous organ sites against several classes of carcinogens. In the current study, we examined the effects of 2 dithiolethiones, 1,2-dithiole-3-thione (D3T) and its structural analogue oltipraz, on DNA adduction induced by the potent mammary carcinogen dibenzo-[a,l]pyrene (DBP) in vivo. Female Sprague-Dawley rats were provided dietary D3T and oltipraz (500 ppm each) for I week followed by a single intragastric dose of DBP (8 micromol/kg body weight) and killed 5 days later. D3T inhibited DBP-DNA adduction from 78% to 82% in all tissues examined, while oltipraz was equally effective in the lung and liver but less effective in the mammary glands, inhibiting DBP-DNA adduction by nearly 60%. These data coupled with their broad anti-tumor specificity support the use of D3T and oltipraz as cancer-preventive agents in clinical trials.
Asunto(s)
Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Benzopirenos/metabolismo , Aductos de ADN , Pirazinas/farmacología , Tionas/farmacología , Tiofenos/farmacología , Animales , Peso Corporal/efectos de los fármacos , Cromatografía , Femenino , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Indole-3-carbinol (I3C) found in various cruciferous vegetables has been shown to exert anti-carcinogenic activity in several target organs. In this study, we have investigated the effects of I3C on cigarette smoke-related lipophilic DNA adduct formation, potentially a key step in chemical carcinogenesis. Female Sprague-Dawley rats were exposed to sidestream cigarette smoke in a whole-body exposure chamber for 6 h per day, 7 days a week for 4 weeks. Control animals received only vehicle while the intervention groups received I3C (1. 36 or 3.40 mmol/kg, b.wt.) daily by gavage starting from 1 week prior to smoke initiation until the end of the experiment. Analysis of tissue DNA by nuclease P1-mediated 32P-postlabeling showed one major and several minor smoke-related adducts in lung, trachea, heart and bladder. The high dose of I3C significantly inhibited the major adducts in lung (#5) and trachea (#3) by 55% each; minor adducts were slightly inhibited (20-40%). The low dose of I3C showed lesser degree of inhibition (30-40%) in both lung and trachea; however, it was found statistically significant in lung only. The major smoke-related adduct in bladder (#2) was strongly inhibited (>65%) by high dose of I3C approaching adduct levels achieved in sham-exposed rats. A small but statistically significant decrease in the smoke-related DNA adduct (#5) in heart tissue was also observed by intervention with high dose I3C. Low levels (30-50 adducts/10(10) nucleotides) of I3C-derived DNA adducts were also found in all the tissues examined although their significance remains unknown. These data show significant inhibition of cigarette smoke-related DNA adducts by I3C, particularly in the lung, trachea, and bladder.
Asunto(s)
Anticarcinógenos/farmacología , Aductos de ADN/efectos de los fármacos , Indoles/farmacología , Humo/efectos adversos , Animales , ADN/efectos de los fármacos , ADN/genética , ADN/metabolismo , Aductos de ADN/metabolismo , Femenino , Corazón/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Miocardio/metabolismo , Plantas Tóxicas , Ratas , Ratas Sprague-Dawley , Nicotiana , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
Dibenzo[a,l]pyrene (DBP), an environmentally significant polycyclic aromatic hydrocarbon (PAH), is one of the most potent carcinogens with greater carcinogenicity in rodent mammary glands and skin than 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene, respectively. In this study, we have examined the formation and persistence of stable DNA adducts in rats administered a carcinogenic intramammillary (i.m.) dose of DBP (0.25 micromol/gland). 32P-post-labeling analysis of mammary epithelial DNA 6 h, and 2, 5 and 14 days post-treatment produced one major (approximately 30%) and at least six minor adducts. Non-target tissue DNA (lung, heart, bladder and pancreas) also showed essentially the same adduct pattern as did mammary DNA, except liver which resulted in four additional adduct spots. The mammary DNA was most adducted (2640 +/- 532 adducts/10(9) nucleotides) on day 5 while the other tissue DNAs had 10- to 65-fold lower adduct levels (lung > liver > heart > bladder > pancreas). Adduct levels continued to increase at all time points examined for all tissues, except mammary tissue which showed a decline ( approximately 40%) on day 14. Chromatographic comparison with adducts formed in vitro by reaction of syn- and anti-DBP-11, 12-diol-13,14-epoxides (DBPDEs) with DNA and individual nucleotides indicated that the in vivo adducts were deoxyadenosine- and deoxyguanosine-derived, formed by interaction with both the anti- and syn-isomers; the adenine-derived adducts comprised 60-75% of the total adducts. However, the liver-specific DNA adducts (nos 8-11) were not derived from any of the DBPDE isomers. Our data show: (i) significantly higher DBP-DNA adduction in mammary tissue as compared with non-target tissues, which is consistent with its mammary carcinogenicity; (ii) adenine is highly reactive towards DBP metabolites as has been observed for many other PAHs; and (iii) the peak binding of DBP with DNA was shifted beyond 14 days for the non-target tissues by i.m. route of exposure.
Asunto(s)
Benzopirenos/toxicidad , Carcinógenos/toxicidad , Aductos de ADN/biosíntesis , Animales , Citocromo P-450 CYP1A1/metabolismo , Femenino , Neoplasias Mamarias Experimentales/inducido químicamente , Ratas , Ratas Sprague-DawleyRESUMEN
Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[a]pyrene (BP) and its bay-region diol epoxide, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a ]pyrene (anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/química , Aductos de ADN/análisis , Aductos de ADN/química , Compuestos Epoxi/química , Benzopirenos/toxicidad , Carcinógenos/toxicidad , Cromatografía en Capa Delgada , ADN/química , Isomerismo , Estructura Molecular , Oligodesoxirribonucleótidos/química , Radioisótopos de FósforoRESUMEN
A significant depletion in the content of glutathione (GSH) and alteration in GSH redox system enzymes were observed in the lung of chrysotile-exposed animals (5 mg) during different developmental stages of asbestosis. In the alveolar macrophages (AM) of exposed animals, the depletion in GSH started from day 1 and reached a maximum at day 16, whereas in lung tissue the maximum depletion was observed when fibrosis has matured. It appears that cellular GSH depletion triggers oxidative stress in the system as observed from increased thiobarbituric acid reactive substance (TBARS) production and alteration in the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD) and glutathione S-transferase (GST), the enzymes regulating oxidative tone. The depletion in GSH was also observed in red blood cells (RBC) of the exposed animals reaching a maximum when fibrosis matured. Thus the observed depletion in GSH, ascorbic acid and alteration in GSH redox system enzymes may be involved in fibrosis and carcinogenesis induced by chrysotile.
Asunto(s)
Asbestos Serpentinas/toxicidad , Asbestosis/etiología , Glutatión/metabolismo , Animales , Asbestosis/metabolismo , Ácido Ascórbico/metabolismo , Femenino , Peroxidación de Lípido , RatasRESUMEN
Exposure to tobacco smoke has been implicated in the increased incidence of cancer and cardiovascular diseases. This report describes various experimental studies in animals that were carried out to determine the ability of cigarette smoke to form DNA adducts and to define chromatographic nature of the major adducts. Tissues from rodents exposed to mainstream or sidestream cigarette smoke in nose-only and whole-body exposure systems, respectively, for different durations were analyzed for DNA adducts by 32P-postlabeling assay. The results showed essentially similar qualitative patterns in various respiratory (lung, trachea, larynx) and non-respiratory (heart, bladder) tissues of smoke-exposed rats. However, adduct pattern in the nasal mucosa was different. The mean total DNA adducts in various tissues expressed as per 1010 nucleotides exhibited the following order: heart (700)>lung (420)>trachea (170)>larynx (150)>bladder (50). Some qualitatively identical adducts were routinely detected in tissues from sham-treated rats but at greatly reduced levels (5- to 25-fold). The levels of lung DNA adducts increased with the duration of exposure up to 23 weeks and returned to control levels 19 weeks after the cessation of exposure. Species-related differences in adduct magnitude and patterns were observed among rats, mice and guinea pigs; mouse being the most sensitive to DNA damage and guinea pig the least sensitive. Whole-body exposure of rats to sidestream cigarette smoke also enhanced the pre-existing DNA adducts by several fold in different tissues. Selective chromatography, and extractability in butanol suggested lipophilic nature of smoke-associated DNA adducts, which were, however, recovered significantly better in nuclease P1 than butanol enrichment procedure. The major smoke-associated adducts were chromatographically different from any of the reference adducts of polycyclic aromatic hydrocarbons (PAHs) co-chromatographed with the smoke DNA samples. Because PAH-DNA adducts are recovered with equal efficiency by the two enrichment procedures, the above observations suggested that smoke-associated adducts are not related to typical PAHs, like benzo[a]pyrene. It is concluded that cigarette smoke increased the levels of pre-existing endogenous DNA adducts (the so-called I-compounds) in animal models and that these adducts are unrelated to those formed by typical PAHs.
Asunto(s)
Aductos de ADN/efectos de los fármacos , Fumar/efectos adversos , Animales , Carcinógenos/toxicidad , Daño del ADN/efectos de los fármacos , Cobayas , Ratones , Especificidad de Órganos , RatasRESUMEN
The present study investigated the effects of dietary oltipraz on cigarette smoke-related lipophilic DNA adduct formation. Female Sprague-Dawley rats were exposed daily to sidestream cigarette smoke in a whole-body exposure chamber 6 h/day for 4 consecutive weeks. One group of rats was maintained on control diet while another group received the same diet supplemented with either a low (167 p.p.m.) or high (500 p.p.m.) dose of oltipraz, starting 1 week prior to initiation of smoke exposure until the end of the experiment. Analysis of lipophilic DNA adducts by the nuclease P1-mediated 32P-post-labeling showed up to five smoke-related adducts. Adduct no. 5 predominated in both the lung and the heart while adduct nos 3 and 2 predominated in the trachea and bladder, respectively. Quantitative analysis revealed that the total adduct level was the highest in lungs (270+/-68 adducts/10(10) nucleotides), followed by trachea (196+/-48 adducts/10(10) nucleotides), heart (141+/-22 adducts/10(10) nucleotides) and bladder (85+/-16 adducts/10(10) nucleotides). High dose oltipraz treatment reduced the adduct levels in lungs and bladder by >60%, while the reduction in lungs in the low-dose group was approximately 35%. In trachea, the effect of low and high dietary oltipraz on smoke DNA adduction was equivocal, while smoke-related DNA adducts in the heart were minimally inhibited by high-dose oltipraz. In a repeat experiment that employed a 3-fold lower dose of cigarette smoke, oltipraz (500 p.p.m.) was found to inhibit the formation of DNA adducts in rat lungs and trachea by 80 and 65%, respectively. These data clearly demonstrate a high efficacy of oltipraz in inhibiting the formation of cigarette smoke-induced DNA adducts in the target tissues.
Asunto(s)
Anticarcinógenos/farmacología , Aductos de ADN/metabolismo , Pirazinas/farmacología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Anticarcinógenos/administración & dosificación , Aductos de ADN/antagonistas & inhibidores , Femenino , Pulmón/metabolismo , Miocardio/metabolismo , Pirazinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tionas , Tiofenos , Tráquea/metabolismo , Vejiga Urinaria/metabolismoRESUMEN
Due to the large and expanding number of potential cancer chemopreventive agents, there is an increasing need for short term tests to study the efficacy and mechanisms of these agents. In this study, we have employed a microsome-mediated test system to study the effect of several suspected chemopreventive agents on the DNA adduct formation capacity of the potent mammary carcinogen, dibenzo[a,l]pyrene (DBP). Bioactivation of DBP by Aroclor 1254-induced rat liver microsomes in the presence of calf thymus DNA (300 microg/ml) resulted in the formation of one major and six other prominent DNA adducts (324 adducts/10(7) nucleotides). These adducts were previously determined to be deoxyadenosine (dA) and deoxyanosine (dG)-derivatives of both anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDE). Intervention with ellagic acid, chlorophyllin, benzyl isocyanate (BIC), oltipraz or genistein (150 microM) strongly diminished DBP-DNA adduction by > or = 75%. Linoleic acid, curcumin and butylated hydroxytoluene (BHT) also significantly inhibited DBP DNA adduction (26-46%) while N-acetylcysteine (NAC) had no effect. Moreover, nonenzymatic studies with anti- and syn-DBPDE isomers revealed that chlorophyllin, ellagic acid, BIC and BHT may be inhibiting DBP-DNA adduction in an enzymatic-independent manner since these agents diminished DBPDE-DNA adduction by 30-75%. Genistein, oltipraz and curcumin did not diminish DBPDE-DNA adduction and therefore most likely require the presence of the microsomal subcellular fraction to inhibit DBP-DNA adduction.
Asunto(s)
Antineoplásicos/farmacología , Benzopirenos/metabolismo , Carcinógenos/metabolismo , Aductos de ADN/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Animales , Benzopirenos/toxicidad , Biotransformación , Carcinógenos/toxicidad , Quimioprevención , Femenino , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/tratamiento farmacológico , RatasRESUMEN
Several observations, including studies from this laboratory, demonstrate that asbestos generates free radicals in the biological system that may play a role in the manifestation of asbestos-related cytotoxicity and carcinogenicity. It has also been demonstrated that iron associated with asbestos plays an important role in the asbestos-mediated generation of reactive oxygen species. Exposure to asbestos leads to degradation of heme proteins such as cytochrome P450-releasing heme in cytosol. Our simulation experiments in the presence of heme show that such asbestos-released heme may increase lipid peroxidation and can cause DNA damage. Further, heme and horseradish peroxidase (HRP) can cause extensive DNA damage in the presence of asbestos and hydrogen peroxide/organic peroxide/hydroperoxides. HRP catalyzes oxidation reactions in a manner similar to that of prostaglandin H synthetase. Iron released from asbestos is only partially responsible for DNA damage. However, our studies indicate that DNA damage mediated by asbestos in vivo may be caused by a combination of effects such as the release and participation of iron, heme, and heme moiety of prostaglandin H synthetase in free radical generation from peroxides and hydroperoxides.
Asunto(s)
Asbesto Crocidolita/toxicidad , Carcinógenos/toxicidad , Daño del ADN/efectos de los fármacos , Hemo/toxicidad , Hemoproteínas/toxicidad , Animales , Bovinos , ADN/química , ADN/efectos de los fármacos , Polvo/efectos adversos , Pulmón/efectos de los fármacos , Pulmón/ultraestructura , Microsomas/efectos de los fármacos , Oxidación-Reducción , Ratas , Especies Reactivas de Oxígeno/fisiología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.
Asunto(s)
Benzopirenos/metabolismo , Aductos de ADN/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Arocloros/toxicidad , Carcinógenos/toxicidad , Cromatografía , Citocromo P-450 CYP1A1/metabolismo , Aductos de ADN/análisis , Inhibidores Enzimáticos/farmacología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Nucleótidos/metabolismo , Ratas , Ratas Sprague-Dawley , beta-naftoflavona/farmacologíaRESUMEN
Dibenzo[a,l]pyrene (DBP) has recently emerged as a potent environmental carcinogen having greater carcinogenicity in the rat mammary epithelial glands than 7,12-dimethylbenz[a]anthracene (DMBA), previously considered to be the most potent mammary carcinogen and benzo[a]pyrene (BP), a ubiquitous environmental carcinogen. Previous studies on the tumor-initiating potential of DBP, DMBA, and BP demonstrated that DBP was 2.5 times more potent in inducing the tumors in mouse skin and rat mammary glands than DMBA; BP was a weak mammary carcinogen in these animals. The present study was designed to investigate if the significantly increased mammary carcinogenicity of DBP over DMBA and BP was related to increased DNA adduction at the target site. Female Sprague-Dawley rats were treated by intramammillary injection with an equimolar dose of 0.25 micromol/gland of DBP, DMBA, and BP at the 3rd, 4th and 5th mammary glands on both sides. 32P-Postlabeling analysis of mammary epithelial DNA of rats treated with DBP produced two major (nos. 3 and 6) and at least 5 minor adducts. DMBA treatment resulted in one major and 4 minor DNA adducts while BP produced one major and two minor adducts. Quantitation of the adduct radioactivity revealed that DNA adduction was 6- and 9-fold greater in DBP-treated animals than in BP- and DMBA-treated animals, respectively. The adduct levels per 10(9) nucleotides in mammary epithelial cells for DBP, BP and DMBA were in the following descending order: 1828 +/- 378, 300 +/- 45 and 207 +/- 72, respectively. Tissue distribution of DNA adducts in non-target organs following DBP treatment showed similar adduct pattern as found in the mammary epithelial cells except the liver, which resulted in 4 additional adduct spots; vehicle-treated tissue DNA processed in parallel did not show any detectable adducts. DMBA- and BP-DNA adduct patterns in various tissues were similar to that found in mammary epithelial cells, however, significant quantitative differences were found; BP-DNA adducts were undetectable in the pancreas and bladder. Quantitation of adduct radioactivity showed a 15- to 60-fold lower DBP-DNA adduction in these tissues than the levels found in the mammary tissue; similarly 5-20 and 30-100 times lower DNA adduction was found following treatment with DMBA and BP, respectively. The significantly increased binding of DBP to the mammary epithelial DNA over BP and DMBA is in concordance with its known higher mutagenicity and tumorigenicity.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/toxicidad , Benzo(a)pireno/toxicidad , Benzopirenos/toxicidad , Carcinógenos/toxicidad , Aductos de ADN/metabolismo , Glándulas Mamarias Animales/efectos de los fármacos , 9,10-Dimetil-1,2-benzantraceno/análogos & derivados , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animales , Benzo(a)pireno/metabolismo , Benzopirenos/metabolismo , Carcinógenos/metabolismo , ADN/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Mutágenos/metabolismo , Mutágenos/toxicidad , Radioisótopos de Fósforo/metabolismo , Ratas , Ratas Sprague-Dawley , Distribución Tisular , Células Tumorales CultivadasRESUMEN
In order to examine the pulmonary toxicity of kerosene oil and its combustion product (soot) in asbestos-exposed rats, various biochemical and chemical parameters were assayed. Treatment of rats with a single intratracheal dose of chrysotile asbestos (5 mg) and kerosene (50 microliters) or its soot (5 mg) in combination led to an increased number of pulmonary alveolar macrophages (PAM), elevated levels of hydrogen peroxide, and thiobarbituric acid-reacting substances, alterations in the activities of primary (glutathione peroxidase and catalase) and secondary (glutathione reductase and glucose-6-phosphate dehydrogenase) endogenous antioxidant enzymes, and depletion in the levels of glutathione in PAM compared to the chrysotile, kerosene, or soot alone. These changes may indicate the generation of oxidative stress in the macrophages. The resulting oxidative stress may be subsequently critical in collapsing the cellular membrane, which may change the cell membrane permeability and may also damage the phagolysosomal membrane, thereby releasing the membrane bound enzymes as indicated by an increased leakage of intracellular acid phosphatase and lactate dehydrogenase. The injury to macrophages may trigger events that lead to lung fibrosis and/or malignancies in the exposed animals. This study may be helpful in understanding the etiology of certain clinical and pathological disorders in the population exposed simultaneously to both asbestos and kerosene or its combustion products.
Asunto(s)
Asbestos Serpentinas/toxicidad , Queroseno/toxicidad , Macrófagos Alveolares/efectos de los fármacos , Animales , Carbono , Sinergismo Farmacológico , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Peróxido de Hidrógeno/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Enfermedades Pulmonares/inducido químicamente , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/metabolismo , Masculino , Estrés Oxidativo , RatasRESUMEN
The glutathione and vitamin E dependent protection of lipid peroxidation in an NADPH (0.4 mM) and chrysotile (500 micrograms/ml) containing system were investigated in vitro in rat lung microsomes. Addition of 1 mM glutathione to the above reaction system containing microsomes supplemented with vitamin E (1 nmol/mg protein) reduced lipid peroxidation. Similar protection by glutathione could be observed in normal unsupplemented microsomes though the degree of protection was less pronounced. Addition of free radical scavengers such as, superoxide dismutase (100 units/ml), catalase (150 units/ml), mannitol (1 mM) and beta-carotene (0.5 mM) to the reaction system showed an insignificant effect on lipid peroxidation. When the reaction was carried out in absence of glutathione, vitamin E content of peroxidizing microsomes decreased rapidly. In this system a concomitant increase in the activity of microsomal glutathione-S-transferase was observed which may serve as an alternative pathway to detoxify lipid peroxides. Addition of glutathione alone to the reaction system prevented both against the loss in vitamin E content and increase in the activity of glutathione-S-transferase. Supplementation of both vitamin E and glutathione was found to be effective in lowering glutathione-S-transferase activity to that of normal basal level. Our results suggest that chrysotile-mediated stimulation of NADPH-dependent lipid peroxidation may be due to hampering of glutathione-dependent protection which may ultimately exhaust membrane bound vitamin E. Our data further suggest that the lung tissue may have an inbuilt mechanism whereby glutathione-S-transferase may be triggered to cope with the excessive production of lipid peroxides.
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Antioxidantes/farmacología , Asbestos Serpentinas/toxicidad , Carcinógenos/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Pulmón/efectos de los fármacos , Animales , Asbestos Serpentinas/administración & dosificación , Catalasa/farmacología , Femenino , Depuradores de Radicales Libres/farmacología , Glutatión/farmacología , Glutatión Transferasa/metabolismo , Pulmón/metabolismo , Manitol/farmacología , Microsomas/efectos de los fármacos , Microsomas/enzimología , NADP/farmacología , Ratas , Superóxido Dismutasa/farmacología , Vitamina E/farmacología , beta Caroteno/farmacologíaRESUMEN
Our previous studies suggested that body fluids such as serum can serve as a novel surrogate for DNA-containing tissues to overcome the major limiting factor of tissue availability in human biomonitoring assessments. We have now examined the detectability of DNA-reactive metabolites (DRMs) in the serum and tissues of male C57BL/6 mice administered up to 10 i.p. injections on alternate days of individual polynuclear aromatic hydrocarbons (PAHs) or a mixture containing 0.01, 0.1, 1.0 and 5.0 mg/kg body wt each of benzo[a]pyrene (BP), cyclopenta[cd]pyrene (CPP) and benzo[k]fluoranthene (BF) in 200 microl sunflower oil. Four hours after the last dose, blood serum was separated and incubated with salmon testes DNA (st-DNA). 32P-Postlabeling of the st-DNA showed essentially the same adduct pattern as found in the tissue DNA, indicating that DRMs of the PAHs used were presumably sequestered and accumulated in the serum proteins. Serum DRMs were detectable at all doses tested following exposure to a mixture of BP, CPP and BF, with varying degrees of detection of the individual PAHs. At doses > or = 1 mg/kg, CPP was more potent than BP and BF in producing serum DRMs as well as tissue DNA adducts. At lower doses, however, BP was more potent. Serum DRMs were enhanced 7-20 times when the animals received 1 mg/kg each of these PAHs in the form of a mixture as compared with individual PAHs, however, tissue DNA adduction was enhanced to a lesser degree (1.5- to 2-fold). Results from these studies suggest that: (i) DNA adduction by a particular carcinogen can be significantly higher when administered in the form of a mixture than administered individually; (ii) DNA-reactive metabolites can be detected by 32P-postlabeling in the serum of animals treated with environmentally significant doses of an artificial carcinogen mixture. This approach has implications for human biomonitoring and offers an advantage because this obviates the need to acquire human tissue DNA, a major obstacle in population biomonitoring studies.
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Carcinógenos/administración & dosificación , Aductos de ADN/análisis , Daño del ADN/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/administración & dosificación , Animales , Carcinógenos/análisis , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales , Humanos , Hígado/química , Pulmón/química , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/química , Hidrocarburos Policíclicos Aromáticos/análisis , Distribución TisularRESUMEN
Asbestos is known to induce oxidative stress in the lung. The consumption of butylated hydroxyanisole (BHA) in preserved food and soft drinks is increasing in the general population, which includes workers in asbestos factories. Because there is no information on the effect of co-exposure to chrysotile and BHA, the time-dependent effects of a single intratracheal dose of chrysotile (1 mg per mouse) and a single ip dose of BHA (350 mg/kg body weight) on various indices of oxidative stress such as lipid peroxidation, hydrogen peroxide generation, glutathione peroxidase (GPX), glutathione reductase (GR), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione (GSH) were followed for up to 14 days. Microsomal lipid peroxidation (as well as that induced by NADPH) was significantly enhanced by BHA in the chrysotile-exposed group. GPX and GR activities in the same group were gradually decreased by BHA. Non-significant modulation of catalase activity by BHA was also noted. BHA induces GSH to a significant extent in lungs exposed with chrysotile. An increase in the G6PDH activity was maximal (19%; P < 0.05) at day 3. The results clearly demonstrate that BHA enhances chrysotile-induced oxidative stress in the lung.
Asunto(s)
Asbestos Serpentinas/toxicidad , Hidroxianisol Butilado/toxicidad , Pulmón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Amianto , Asbestos Serpentinas/administración & dosificación , Asbestos Serpentinas/efectos adversos , Hidroxianisol Butilado/administración & dosificación , Hidroxianisol Butilado/efectos adversos , Catalasa/metabolismo , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Conservación de Alimentos , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Inyecciones Intraperitoneales , Intubación Intratraqueal , Peroxidación de Lípido/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Microsomas/efectos de los fármacos , Microsomas/enzimología , Exposición ProfesionalRESUMEN
This article reports the effect of coexposure to Indian chrysotile asbestos (5 mg/rat) and kerosene soot (5 mg/rat) on the pulmonary phase I and phase II drug-metabolizing enzymes 1, 4, 8, 16, 30, 90, and 150 days after a single intratracheal inoculation. Exposure to soot resulted in a significant induction of the pulmonary microsomal cytochrome P450 and the activity of dependent monooxygenase, benzo(a)pyrene (B[a]P) hydroxylase, and epoxide hydrase at all time intervals. On the other hand, the cytosolic glutathione S-transferase (GST) activity was induced at days 1, 4, 8, 16, and 30 after exposure, followed by inhibition in the enzyme activity. In contrast, chrysotile exposure depleted cytochrome P450, B[a]P hydroxylase, epoxide hydrase, and GST at initial stages, while all these parameters except GST were induced at later stages. However, coexposure to chrysotile and soot led to a significant inhibition in the cytochrome P450 levels, activities of B[a]P hydroxylase, epoxide hydrase, and GST at initial stages of exposure. At advanced stages, however, an additional increase in cytochrome P450, B[a]P hydroxylase, and epoxide hydrase but a decrease in GST was observed. These results clearly show that the intratracheal coexposure to high levels of asbestos and kerosene soot alters the metabolic activity of the lung, which is turn may retain toxins in the system for a longer period, resulting in adverse pathological disorders.