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Archives of Toxicology (v.78, #3)
Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP by Holger M. Koch; Hermann M. Bolt; Jürgen Angerer (pp. 123-130).
Human metabolism of di(2-ethylhexyl)phthalate (DEHP) was studied after a single oral dose of 48.1 mg to a male volunteer. To avoid interference by background exposure the D4-ring-labelled DEHP analogue was dosed. Excretion of three metabolites, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP), was monitored for 44 h in urine and for 8 h in serum. Peak concentrations of all metabolites were found in serum after 2 h and in urine after 2 h (MEHP) and after 4 h (5OH-MEHP and 5oxo-MEHP). While the major metabolite in serum was MEHP, the major metabolite in urine was 5OH-MEHP, followed by 5oxo-MEHP and MEHP. Excretion in urine followed a multi-phase elimination model. After an absorption and distribution phase of 4 to 8 h, half-life times of excretion in the first elimination phase were approximately 2 h with slightly higher half-life times for 5OH- and 5oxo-MEHP. Half-life times in the second phase—beginning 14 to 18 h post dose—were 5 h for MEHP and 10 h for 5OH-MEHP and 5oxo-MEHP. In the time window 36 to 44 h, no decrease in excreted concentrations of 5OH- and 5oxo-MEHP was observed. In the first elimination phase (8 to 14 h post dose), mean excretion ratios of MEHP to 5oxo-MEHP and MEHP to 5OH-MEHP were 1 to 1.8 and 1 to 3.1. In the second elimination phase up to 24 h post dose mean excretion ratios of MEHP to 5oxo-MEHP to 5OH-MEHP were 1 to 5.0 to 9.3. The excretion ratio of 5OH-MEHP to 5oxo-MEHP remained constant through time at 1.7 in the mean. After 44 h, 47% of the DEHP dose was excreted in urine, comprising MEHP (7.3%), 5OH-MEHP (24.7%) and 5oxo-MEHP (14.9%).
Keywords: Metabolism; Urine; Serum; Di(2-ethylhexyl)phthalate (DEHP); Mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP); Mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP); Mono(2-ethylhexyl)phthalate (MEHP)
Age-related changes in the protein and mRNA levels of CYP2E1 and CYP3A isoforms as well as in their hepatic activities in Wistar rats. What role for oxidative stress? by Valérie Wauthier; Roger K. Verbeeck; Pedro Buc Calderon (pp. 131-138).
Drug biotransformation and its therapeutic effect may be modified during ageing. Among different causative factors of ageing, the impairment of normal cellular functions by free radicals has been evoked as playing a critical role. The effect of age on the expression and activity of CYP2E1 and CYP3A was investigated in male Wistar rats of 3, 8, 11 and 18 months old. The total cytochrome P450 as well as the expression and the activity (midazolam oxidation) of CYP3A isoforms did not change until 18 months of age. Chlorzoxazone hydroxylation (CYP2E1 activity) increased from 3 to 8 months, remained constant between 8 and 11 months and then progressively decreased until 18 months. Interestingly, CYP2E1 microsomal protein followed the same enzyme activity profile from 3 to 8 months, but remained constant thereafter. The level of CYP2E1 mRNA did not change over the whole period. While the amount of proteins did not change after 8 months, their functionality may be affected by oxidative stress (increase in thiobarbituric acid reactive substances, decrease in reduced glutathione level). However, no changes in carbonyl protein content were observed. The decrease in CYP2E1 activity in rats after 11 months is most probably due to post-translational modifications of CYP2E1 proteins. Indeed, it may be correlated with an accumulation of oxidative damage. Since no change was observed in CYP3A activity or in their protein and mRNA content, it seems that such isoforms should be less affected by oxidative stress.
Keywords: Cytochrome P450; Ageing; Oxidative stress; Proteasome
Free radical generating agents lead to the rapid progression of benign skin tumors to carcinoma in iron-overloaded mice by Gayatri Bhasin; Hina Kauser; Mohammad Athar (pp. 139-146).
Free radical generating compounds have been shown to enhance the malignant conversion of papillomas to carcinomas in mouse skin, and iron has been shown to participate in free radical generating reactions. In the present study, we investigated whether iron can play a role in the malignant conversion of papillomas to carcinomas. Skin tumors were chemically induced in female Swiss albino mice using a standard two-stage initiation-promotion protocol. Topical application of 12-O-tetradecanoyl phorbol 13-acetate (TPA), benzoyl peroxide (BPO), H2O2 and cumene hydroperoxide (COOH) to these tumor-bearing mice increased the rate of malignant conversion. To evaluate the effect of iron-overload on the conversion of benign skin papillomas to carcinomas, the animals were pre-treated with 1.0 mg Fe per mouse for 15 days before they received TPA or free radical generating compounds. The number of carcinomas and the percent incidence of carcinomas were recorded weekly. The rate of malignant conversion was higher in iron-overloaded mice as compared with non-iron-overloaded mice. The ability of iron-overload in enhancing the malignant conversion was in the order TPA2O22O2)-mediated malignant transformation was also enhanced effectively by iron. This may be because a combination of iron accessibility and H2O2 results in the formation of the very reactive hydroxyl radical via the Fenton reaction, which can cause DNA damage. Besides this, the cutaneous iron levels were also higher in iron-overloaded mice as compared with non-iron-overloaded mice. Histopathological sections of tumors also showed a higher degree of keratinization and pearl formation in iron-overloaded animals. Thus, we observe that in iron-overloaded animals, free radical generating agents bring about the rapid progression of benign mouse skin papillomas to carcinomas.
Keywords: Free radicals; Iron-overload; Tumor progression; Benign papilloma; Carcinoma
Cellular and molecular studies on cisplatin-induced apoptotic cell death in rat kidney by David Sheikh-Hamad; William Cacini; Arthur R. Buckley; Jorge Isaac; Luan D. Truong; Chun Chui Tsao; Bellamkonda K. Kishore (pp. 147-155).
Using morphological and molecular approaches, we characterized cisplatin-induced cell necrosis and apoptosis in rat kidney. Male Sprague-Dawley rats (n=5 per group) received a single intraperitoneal injection of either cisplatin (5 mg/kg) or saline, and were killed on day 5. Functionally, cisplatin-treated rats developed polyuric acute renal failure. Morphologically, kidneys of cisplatin-treated rats showed overt tubular necrosis associated with apoptosis in the corticomedullary junction. Cell necrosis was segment-specific and was distributed in radial fashion at the corticomedullary junction. The apoptosis was limited to discrete cells in apparently intact tubules in the vicinity of the necrosed tubules. The apoptotic changes were confirmed by TUNEL (TdT-mediated deoxyuridine triphosphate nick-end labeling) and staining for cleaved caspase-3. Analysis of outer medullary tissue for apoptosis-related molecules by RNase protection assay revealed a significant increase in the expression of pro-apoptotic mRNAs (caspases 1, 2, and 8, and Bax) in cisplatin-treated rats. On the other hand, the expression of mRNA for the anti-apoptotic Bcl-2 did not change, resulting in a decrease in relative ratio of Bcl-2/Bax, and thus favoring apoptosis. The above changes were paralleled by a marked increase in caspase-3 precursor, the executioner protease. Furthermore, these pro-apoptotic molecular changes were associated with a 3-fold increase in the activity of JNK1 in the outer medulla, but not in the cortex, of cisplatin-treated rat kidneys, localizing to the site of maximal apoptosis. Upregulation of JNK1 activity in the outer medulla was not accompanied by changes in the activities of ERK or p38 kinase. In conclusion, these data suggest that cisplatin-induced apoptotic cell death in native kidney may be mediated by cooperative activation of the JNK1 pathway and Bax in the outer medulla.
Keywords: Cisplatin; Acute renal failure; Apoptosis; Caspases; MAP kinases
Protective effect of topical iodine containing anti-inflammatory drugs against sulfur mustard-induced skin lesions by Uri Wormser; Amnon Sintov; Berta Brodsky; Robert P. Casillas; Abraham Nyska (pp. 156-166).
Previous studies have shown the antidotal efficacy of topical iodine at 15 and 30 min post-exposure to sulfur mustard (SM). Here we demonstrate efficacy at longer intervals (20, 30, 45, and 60 min, respectively, for data) using an improved topical povidone-iodine preparation termed N66, which contains steroidal and non-steroidal anti-inflammatory agents. In the mouse, N66 reduced severity of ear edema by 43, 47, 44, and 36%; ear epidermal ulceration by 74, 58, 45, and 58%; and epidermal necrosis by 54, 34, 26, and 31% at the respective time points. A similar effect was observed with encrustation. The healing marker, grade of acanthotic area, showed dramatic increases of 39.6-, 25.3-, 20.9-, and 22-fold. Severity of the dermal parameters, acute inflammation and dermal necrosis, was reduced by 63, 34, 34, and 38% and 80, 54, 54, and 59%, respectively. In guinea pig skin, topical treatment with N66 45 min post-exposure reduced the SM-induced ulceration area by 75%. The histological parameters subepidermal microblister formation, epidermal ulceration, epidermal necrosis, and encrustation were reduced by 63, 61, 41, and 41%, respectively. The healing marker, grade of acanthotic area, was elevated by 73%. N66 induced a statistically significant reduction in two dermal markers for tissue damage: acute inflammation (33%) and dermal necrosis (48%). Reduced skin damage was also observed in areas adjacent the treated sites. The pharmacologically active components of N66 showed additive effect. These findings suggest that the povidone-iodine preparation combined with anti-inflammatory agents functions as a potent antidote against skin lesions induced by SM at relatively long intervals between exposure and treatment.
Keywords: Sulfur mustard; Mustard gas; Skin toxicity; Dermatotoxicity; Iodine; Anti-inflammatory agents
The tumorigenic characteristics of Lime-Piper betel quid-transformed JB6 cells by Ming-Hsun Lin; Chau-Jong Wang; Hui-Pei Huang; Ming-Yung Chou; Fen-Pi Chou (pp. 167-173).
Betel quid chewing is a general oral habit in Taiwan, India, southeastern Asian and South Africa with or without the additive of tobacco, alcohol or lime. In this study, the tumor-promoting neoplastic transformation effect of Lime-Piper betel quid (LPB) was examined on JB6 cells. The treatment of LPB at a high dose (1.0 mg/ml) for over 5 days or at lower doses (0.1, 0.5 mg/ml) for over 15 days induced the formation of transformed foci. The transformed cells showed the characteristics of colony formation in soft agar, higher growth rate and multilayer on culture dish. A two-fold induction of the protein levels of c-fos and c-jun proto-oncogenes was observed in the cells from the 50th passage (Cl1/p50, Cl2/p50 and Cl3/p50), suggesting that LPB-transformed cells were oncogenic. In addition, the LPB-transformed cells possessed an elevated level of c-Myc and an increased cell population distributed in the S phase of the cell cycle. These results demonstrated the promotion effect of LPB and indicate that it could be a tumor promoter.
Keywords: Promotion effect; Lime-piper betel quid; Proto-oncogene; Oncogenic
32P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products by Hanna L. Eriksson; Magnus Zeisig; Lars-Gösta Ekström; Lennart Möller (pp. 174-181).
The carcinogenicity of petroleum products is mainly due to their content of polycyclic aromatic compounds (PACs). These compounds may be activated metabolically and react with DNA to form DNA adducts, which is a critical event in the initiation of cancer. One of the most common techniques for analyzing DNA adducts is 32P-postlabeling. The chromatographic method often used has been 32P-TLC (thin-layer chromatography), but the more recently developed 32P-HPLC (high-performance liquid chromatography) method has shown advantages. The aim of this study was to test the hypothesis that the 32P-HPLC method has a better ability of detecting DNA adducts derived from petroleum products than 32P-TLC. It was found that some DNA adducts migrated from the application point in 32P-TLC in such a way that it is doubtful if they could be detected and quantified properly. It was also found that, when using 32P-HPLC, it is possible to use the same protocol for substances with a wide variety of DNA adduct forming potential, whereas 32P-TLC needs to be optimized regarding time of exposure and/or the amount of DNA applied. Further, a pattern of recognition in 32P-HPLC enables a selective assessment of DNA adducts derived from complex mixtures whereas 32P-TLC is very limited when analyzing complex mixtures due to poor resolution. With more knowledge about the properties of the most mutagenic DNA adducts in HPLC, it could be possible to know also which pattern corresponds to a mutagenic or carcinogenic oil. Consequently, 32P-HPLC is a good alternative when assessing the genotoxicity of petroleum products.
Keywords: Petroleum; DNA adducts; 32P-Postlabeling; 32P-HPLC; Polycyclic aromatic compounds