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Testicular toxicity of dibromoacetonitrile and possible protection by tertiary butylhydroquinone
Pharmacological Research 47 (2003) 509–515
Testicular toxicity of dibromoacetonitrile and possible protection by tertiary butylhydroquinone Mohamed H. Abdel-Wahab∗ Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Accepted 21 January 2003
Abstract Dibromoacetonitrile (DBAN) is known to be water disinfectant by-product. Its broad-spectrum toxicity in different test systems in vivo and in vitro has been reported. Oxidative damage induced by DBAN may be partially responsible for its toxicity. Herein, the ability of DBAN to induce oxidative stress in mouse testis and possible protective effect of an antioxidant tertiary butylhydroquinone (TBHQ) were addressed. Male albino mice were injected with a single dose of DBAN (50 mg/kg i.p.), and killed after 3 h of treatment. Control animals received 10 ml/kg body weight i.p. of the vehicle DMSO. In both experiments, cauda epididymis were dissected and sperm count and motility were investigated. Also, testicular activity of lactic dehydrogenase-x (LDH-x) isozyme and histopathological changes were examined. Furthermore, testicular content of malonyldialdehyde (MDA) and reduced glutathione (GSH) were determined. A single i.p. dose of DBAN caused decrease in sperm count and motility to approximately 88 and 84%, respectively, compared with control animals. A 46% decrease in testicular activity of LDH-x, compared with control animals, was observed. A significant accumulation of MDA in DBAN-treated animals was increased to 99% while testicular content of GSH was decreased by 56% compared to control animals. Compared to DBAN-treated animals, treatment with TBHQ (100 mg/kg p.o.) prior exposure to DBAN showed a remarkable degree of protection as indicated by enhancement of sperm count and motility, testicular activity of LDH-x, and GSH. Accumulation of testicular content of MDA significantly decreased following TBHQ treatment compared to DBAN-treated animals. In conclusion, results presented here indicate that DBAN is capable to induce oxidative stress in mouse testis. TBHQ may play a protective role against DBAN-induced testicular cellular damage. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Oxidative stress; Testes; Dibromoacetonitrile; Tertiary butylhydroquinone; Mice
1. Introduction Chlorination of drinking water results in the formation of a variety of hazardous by-products including halogenated acetonitriles (HANs). These by-products are formed as a consequence of the interaction between chlorine and organic impurities in water [30]. Inclusion of bromine in aqueous solutions of chlorine results in preferential substitution of bromine into organic substrates present in water [15]. It has been reported that consumption of chlorinated water could also result in formation of HAN in vivo [45]. HANs have been used as insecticides for stored grains [17], biological growth inhibitors in cooling towers [42] and as common laboratory chemicals [8]. Besides the contaminated drinking water [57], HANs have been detected as environmental hazards, following ingestion of food products which leached by HAN monomers ∗ Tel.:
+20-202-4724450/4724212. E-mail address: [email protected] (M.H. Abdel-Wahab).
from packing materials [16,56], inhalation of cigarette smoke [11], car exhaust [35] and industrial dust in the work place [62,64]. Epidemiological studies have suggested an association between exposure to chlorinated water and the occurrence of gastrointestinal and urinary tract cancers [23,40,48]. Numerous toxicological effects of HAN have been reported. These include mutagenic [20], carcinogenic [14,19,43,53] and embryotoxic [60] effects. Also, testicular adverse effects of HAN and acrylonitrile have been reported [63]. Induction of oxidative damage by these compound and their ability to release cyanide have been associated with the vast majority of reported toxicities [4,32,33]. Dibromoacetonitrile (DBAN, Br2 –CH–CN) is one of the member of HAN group which also exhibits a wide range of toxicity in a manner similar to that described for HAN. DBAN-induced oxidative stress, in different test systems, has been reported by many investigators [3,65]. Although its broad spectrum toxicity in vivo and in vitro has been addressed, its testicular toxicity has not been fully explored.
1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00039-2
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Butylated hydroxyanisole (BHA) and its metabolite tertiary butylhydroquinone (TBHQ) are widely used as food preservatives, probably due to their powerful antioxidant potential [1,2,49,51,55]. It has been reported that, BHA and TBHQ protect animals against radiation and the acute toxicity of various xenobiotics and mutagens [3,4,31,34,67]. Dietary administration of these compounds also leads to the protection against various carcinogens, presumably through the induction of many phase II detoxifying enzymes such as epoxide hydrolases [7,9,12,47], glutathione S-transferases [8,46] and glucuronosyltransferases [18], as well as through the inhibition of cytochrome P-450 monooxygenase [18]. Biochemical and genetic studies show that the induction of these detoxifying enzymes is primarily due to the transcriptional activation of genes and is regulated by an enhancer, called antioxidant-responsive element or electrophile-responsive element [26–28,39,58,59]. Since DBAN has been shown to induce oxidative stress in different test systems; and growing evidences have indicated the association between oxidative stress and male reproductive impairment [5], the present experiment was designed to investigate the potential testicular toxicity of DBAN in male albino mice. Also, the antioxidant potential of TBHQ to modulate the toxicity was examined as an attempt to explain the mechanism of DBAN-induced testicular toxicity.
2. Materials and methods 2.1. Chemicals DBAN was purchased from Aldrich Chemical Co. (Milwaukee, WI). TBHQ was obtained from Aldrich–Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest quality and commercially available. 2.2. Animals Eighteen adult male albino mice, weighing 18–20 g, were obtained from our local animal facility at Al-Azhar University. Mice were maintained in our laboratory under standard laboratory conditions (12 h light:12 h dark, 25 ± 2 ◦ C) with free access to food and water. Animals were divided equally into three groups of six animals each. Control animals received an i.p. injection of DMSO at dose of 10 ml kg−1 once. Six animals received an i.p. injection of DBAN at a dose of 50 mg kg−1 once. The third group of mice received multiple i.p. doses of TBHQ (100 mg kg−1 for 5 consecutive days) prior to exposure to DBAN. 2.3. Methods 2.3.1. Assessment of sperm count and motility Sperm count and motility were determined according to the method of Anderson et al. [6]. Spermatozoa were ob-
tained by making small cuts in the two cauda epididymis from each mouse. The contents of the epididymal spermatozoa were suspended carefully in 20 ml Krebs–Ringer bicarbonate buffer prewarmed to 37 ◦ C. Approximately, 10 l of the suspension was transferred to each chamber of a Neubauer hemocytometer. Over-filling or under-filling was avoided. Separate counts for motile and non-motile sperms in each chamber were taken. If the variation between the chambers exceeds 10%, the whole estimation was repeated. 2.3.2. LDH-x enzyme activity LDH-x activity was assessed according to the method of Cheever et al. [13]. The testes were homogenized in 0.25 M sucrose solution (1:3, w/v) for 1 min. The homogenates were centrifuged at 10,000 × g for 30 min and the supernatant was passed through 0.45-m pore-size acrodisc (Gelman Sciences, Inc., Ann Arbor, MI). The filtrate was used as LDH-x enzyme preparation. Quartz cuvettes containing 3 ml of the specific enzyme substrate were incubated with 20 l of the enzyme preparation for 10 min. The temperature of the reaction mixture was kept at 30 ◦ C and the change in absorbance was measured photometerically at 340 nm. 2.3.3. Determination of reduced glutathione (GSH) Testicular GSH content was determined photometerically following the method of Sedlak and Lindsay [61]. Samples were prepared for non-protein fraction by mixing equal volumes of cooled 10% trichloroacetic acid solution with testicular homogenate. The protein precipitate was removed by centrifugation at 10,000 × g at 4 ◦ C for 10 min. Sulfhydryl concentration was measured, at 412 nm, after addition of 5,5 -dithio-bis (2-nitrobenzoic acid) to the tissue supernatant in the presence of phosphate buffer [22,36]. 2.3.4. Determination of lipid peroxidation Lipid peroxidation was evaluated as malonyldialdehyde (MDA) production by determination of the tissue content of thiobarbituric acid (TBA) reactive substances (TBARSs) as described by Uchiyama and Mihara [66]. Stock solution of 1,1,3,3-tetraethoxypropane in water was used to produce different concentrations of MDA, these solutions were used as TBARS standards. TBA and 1% H3 PO4 were added to homogenates and heated to boiling for 45 min. TBA adducts were extracted with n-butanol. The TBARS concentrations were determined spectrophotometerically utilizing the differences in optical density (OD) at 535 and 520 nm and using the molar absorption constant of 56 × 105 M−1 cm−1 . 2.3.5. Testes histopathology [37,63] For qualitative analysis of testicular histology, one testis from each animal was fixed in Bouin’s fluid for ∼18 h. The fixed testes were embedded in paraffin wax and blocks were prepared. Testicular sections were collected from each testis at 5 m thickness using a rotary microtome and stained with
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haematoxylin and eosin. Histologic examination was performed using light microscopy. Microphotographs were prepared under low and high magnification.
Table 3 Effect of DBAN and TBHQ plus DBAN on the testicular content of GSH and MDA on mice Parameters
2.3.6. Data analysis The GRAPHPAD INPLOT (ISI Software, Philadelphia, PA) computer program was used to plot collected data. Data were expressed as means ± S.E.M. Assessment of the results was performed using one-way ANOVA procedure followed by Bonferroni multiple comparison test using software GRAPHPAD INSTAT (ISI Software). The 0.05 level of probability was used as the criterion for significance.
3. Results The results of sperm count and motility following DBAN and TBHQ plus DBAN are listed in Table 1. I.p. injection of DBAN (50 mg kg−1 , once) caused significant decrease in sperm count and motility by 88 and 84%, respectively, compared with control mice. Treatment of the animals with TBHQ (100 mg kg−1 per day × 5 p.o.) prior to DBAN showed significant decrease of sperm count and motility by 19 and 26%, respectively, compared with DMSO-treated mice. Upon comparing TBHQ-treated mice with DBAN-treated once, TBHQ was shown to increase sperm count and motility by 564 and 372%, respectively. The effect of DBAN and TBHQ plus DBAN on testicular LDH-x isoenzyme activity is given in Table 2. I.p. injection of DBAN caused significant decrease in the
Table 1 Effect of DBAN and TBHQ plus DBAN on sperm count and motility on mice Parameters
Groups Control group
Sperm count (million) % of sperm motility
DBAN-treated group
TBHQ + DBANtreated group
46.3 ± 2.35
5.67 ± 2.36a
37.67 ± 1.33a,b
90.0 ± 1.39
14.17 ± 4.55a
66.83 ± 3.23a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group. Table 2 Effect of DBAN and TBHQ plus DBAN on LDH-x activity on mice Parameter
LDH-x (U/g)
Groups Control group
DBAN-treated group
TBHQ + DBANtreated group
93.4 ± 4.8
50.0 ± 3.6a
115 ± 7.5a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group.
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Groups Control group
GSH (mol/g protein) MDA (nmol/g protein)
1.85 ± 0.05
DBAN-treated group 0.82 ± 0.095a,b
83.00 ± 5.1 165.00 ± 9.32a,b
TBHQ + DBANtreated group 1.6 ± 0.06 115.0 ± 7.5a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group.
testicular activity of LDH-x by 46% compared with control group. Treatment of mice with TBHQ prior to DBAN caused significant increase in the activity of LDH-x by 23% compared with vehicle-treated animals. Pretreatment of DBAN-treated mice with TBHQ significantly increased the isoenzyme activity by 130% compared with DBAN-treated mice. Concerning the study of the mechanism of testicular toxicity induced by DBAN, the results are shown in Table 3. I.p. injection of DBAN showed a significant decrease in the testicular content of GSH by 56% compared with respective control mice. On the other hand, testicular content of MDA significantly increased by 99% in DBAN-treated animals compared with control. Treatment of the animals with TBHQ prior to DBAN showed significant protective effect as evidenced by normalization of GSH content and mitigation of MDA content. 3.1. Testis histopathology Histopathological examination showed that i.p. injection of DBAN (50 mg kg−1 once) caused marked pathological changes in mice testes (Fig. 1) compared to normal animals (Fig. 3). In the DBAN-treated group, the main damage was found in the seminiferous tubules in the form of oedema patches, pyknosis of spermatogonia and spermocytes, reduction in spermatogonia layers with presence of empty spaces and many bizzar cells. Rupture of basement membrane, swelling and some completely degenerated cells, inhibition of the process of spermatogenesis, presence of giant multinucleated cells and breakdown of the nuclear material (karyolysis) in most cells were observed. Also, degenerated cells were delineated in the seminiferous tubules. Congestion of blood vessels and haemorrhage in the interstitial tissue were noticed. Pretreatment of DBAN-treated animals with TBHQ mitigate some of the previous changes as shown in Fig. 2. This mitigation was in the form of somewhat irregular distribution of the spermatogonic series, basement membrane is slightly affected. Necrotic and karyolysis were less encountered.
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Fig. 1. Pyknosis of spermatids and spermatocytes, oedema, haemorrhage and karyolysis. Magnification: 40×.
Fig. 2. Mitigation of seminiferous tubule of testes. Magnification: 40×.
Fig. 3. Normal seminiferous tubules of testes and presence of spermatogonia. Magnification: 40×.
4. Discussion DBAN, a member of HANs, is produced as a result of chlorination of drinking water. Little information about its potential testicular toxicity as water pollution is available. Hence, this study was designed to investigate the potential testicular toxicity of DBAN and mechanism underlying the
toxicity. Also, possible protective effect of TBHQ, an antioxidant agent, was investigated. The results of the present study showed that single i.p. administration of DBAN in mice significantly decreases the epididymal sperm count and motility compared with control animals. Also DBAN causes histopathological changes in the testis of mice. This is in accordance with a similar
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work of Tandon et al. [63], who reported that exposure of mice to vinyl cyanide (acrylonitrile, VCN) induced damage to the seminiferous epithelium as evidenced by decrease of sperm count and motility. The similarity between our study and the study of Tandon et al. [63] is the release of cyanide ions from both DBAN and VCN metabolism. Cyanide, in turn, interferes with energy production processes required for sperm vitality and motility [24,38]. Also, the testicular activity of LDH-x significantly decreases in DBAN-treated animals. This is in harmony with the study of Abdel Naim [1], who reported that treatment of rats with acrylonitrile inhibited LDH-x activity. This change in LDH-x activity together with the result of sperm count and motility as well as histopathological changes are of valuable index for the testicular toxicity [25]. The mechanism underlying the testicular toxicity of DBAN is not fully explained. However, Ahmed et al. [3] reported that DBAN induced a significant oxidative stress in rat gastric tissues as evidenced by GSH-depletion and GST inhibition. Moreover, induction of oxidative stress was associated with the toxicity of structurally related compounds such as chloroacetonitrile (CAN) [32] and acrylonitrile [4,33]. Our results indicated that i.p. injection of DBAN in mice significantly decreases the testicular content of GSH and increases the content of MDA. This indicates that DBAN induces oxidative stress in testis of mice. This is in the same lines of the study of Jacob et al. [32], Ahmed et al. [4] and Jiang et al. [33]. Several studies have reported the implication of free radicals such as hydroxyl radical and superoxide anion in the testicular toxicity of cyanide-containing compounds such as acrylonitrile [29,41,54]. The formation of reactive oxygen species (OH• , O•− and H2 O2 ) is done through bioactivation of cyanide-containing compounds (VCN, DBAN, CAN) by hepatic microsomal enzymes such as cytochrome-P450 or peroxidases leading to liberation of CN ions which interact with certain compounds inducing free radicals formation [1,2,7,42]. In an attempt to mitigate the testicular toxicity induced by DBAN, we tested the hepatic phase II enzymes induced by TBHQ. The present results showed that, oral administration of TBHQ to the mice prior to DBAN, attenuates the testicular toxicity parameters and mitigates the testicular histopathological changes. This attenuation is due to the potential antioxidant activity of TBHQ [49,52]. Also, the ability of TBHQ to induce hepatic enzymes may play a role [10,44]. TBHQ ameliorates the testicular content of GSH in this study. This may be due to the upregulation of GSH synthesis by TBHQ [21]. The stronger radical scavenger effect of TBHQ may be responsible for the decreased testicular content of MDA [50]. In summary, DBAN has potential testicular toxicity as evidenced by decreased sperm count and motility as well as LDH-x activity. This toxicity was through oxidative stress induction as showed by increased testicular content of MDA and decreased GSH content. The toxicity of DBAN on the testis is mitigated by TBHQ.
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References [1] Abdel Naim AB. Studies on the mechanism of testicular toxicity induced by acrylonitrile. Ph.D. Thesis, Al-Azhar University, Cairo, Egypt; 1995. [2] Ahmed AE, Farooqui MYH, Trieff NM. Nitriles. In: Anders MW, editor. Bioactivation of foreign compounds. Orlando, FL: Academic Press, Inc. p. 485–518. [3] Ahmed AE, Hussein GI, Loh JP, Abdel-Rahman SZ. Studies on the mechanism of haloacetonitrile-induced gastrointestinal toxicity: interaction of dibromoacetonitrile with glutathione and glutathione-S-transferase in rats. J Biochem Toxicol 1991;6(2):115– 21. [4] Ahmed AE, Nouraleen AM, Abdel-Rahman SZ, Rajarman S. Role of glutathione modulation in acrylonitrile-induced gastric DNA damage in rats. Arch Toxicol 1996;70(10):620–7. [5] Ahotupa M, Huhtaniemi I. Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally criptorchid rat testis. Biol Reprod 1992;46:1114–8. [6] Anderson RA, Willis BR, Oswald C, Zaneyeld LJD. Ethanol induced male infertility: impairment of spermatozoa. J Pharmacol Exp Ther 1983;225(2):479–86. [7] Aust SD, Chignell CF, Bxay TM, Kalyanaraman B, Masson RP. Contemporary issues in toxicology. Free radical in toxicology. Toxicol Appl Pharmacol 1993;120:168–78. [8] Barcelo D, Maris FA, Geedink Rb, Frei RW, de Jong GJ, Brinkman UA. Comparison between positive, negative and chloride-enhanced negative chemical ionization of organophosphorus pesticides in on-line liquid chromatography–mass spectrometry. J Chromatogr 1987;8:65–76. [9] Benson AM, Batzinger RP, Ou S-YL, Bueding E, Cha Y-N, Talalay P. Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Cancer Res 1978;38:4486–95. [10] Bock KW, Eckle T, Ouzzine M, Fournel-Gigleux S. Coordinator induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the optical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells. Biochem Pharmacol 2000;59:467–70. [11] Byrd GD, Fowler KW, Hicks RD, Lovette ME, Bovgerding MF. Isotop dilution gas chromatography–mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke. J Chromatogr 1990;503:359–68. [12] Cha YN, Martz F, Beuding E. Enhancement of liver microsome epoxide hydratase activity in rodents by treatment with 2(3)-tert-butyl-4-hydroxyanisole. Cancer Res 1978;38:4496–8. [13] Cheever KL, Weigel WW, Rechards DE, Lal JB, Plotnick HB. Testicular effect of bis (2-methoxyethyl) ether in the adult male rat. Toxicol Ind Health 1989;5(6):1099–109. [14] Clayson DB, Iverson F, Nera EA, Lok E. The significance of induced forestomach tumors. Annu Rev Pharmacol Toxicol 1990;30:441–63. [15] Coleman WE, Munch JW, Kaylor WH, Streicher RP, Ringhand HP, Meier JR. GC/MS analysis of mutagenic extracts of aqueous chlorinated humic acid: comparison of the by-products to drinking water contaminants. Environ Sci Technol 1984;18:674–81. [16] Conacher HB, Page BD, Ryan JJ. Industrial chemical contaminants of foods. Food Addit Contam 1993;10:129–43. [17] Cotton RT, Walkden HH. The role of sorption in the fumigation of stored grain and cereal products. J Kans Entomol Soc 1968;17:98. [18] Cummings SW, Prough RA. Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions. J Biol Chem 1983;258:12315–9. [19] Daniel FB, Schenck KM, Mattox JK, Lin EL, Haas DL, Pereira MA. Genotoxic properties of haloacetonitriles: drinking water by-products of chlorine disinfection. Fundam Appl Toxicol 1986;6(3):447–53. [20] Duverger-van bogaert M, Lambotte-Vandepaer M, De Meester C, Rollmann B, Poncelet F, Mercier M. Effect of several factors on the
514
[21]
[22] [23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36] [37]
[38]
[39]
[40] [41]
[42]
M.H. Abdel-Wahab / Pharmacological Research 47 (2003) 509–515 liver extract mediated mutagenicity of acrylonitrile and identification of four new in vitro metabolites. Toxicol Lett 1981;7:311–9. Eftekhar EP, Arne H, Bernhord HJ. Thioredoxin reductase and glutathione synthesis is regulated by TBHQ in cortical astrocytes but not in cortical neurons. GLIA 2000;31:241–8. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;74:214–26. Flaten TP. Chlorination of drinking water and cancer incidence in Norway. Int J Epidemiol 1992;21:6–15. Folgero T, bertheussen K, Lindal S, Toybergsen T, Oian P. Mitochondirial disease and reduced sperm motility. Hum Reprod 1993;8(11):1863–8. Foster PMD, Blackburn DM, Moore RB, Lloyd SC. Testicular toxicity of 2-methoxyacetaldehyde, a possible metabolite of ethylene glycol monomethyl ether in the rat. Toxicol Lett 1986;32:289–314. Friling RS, Bensimon A, Tichauer Y, Daniel V. Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proc Natl Acad Sci USA 1990;87:6258–62. Galloway DC, McLellan LT. Inducible expression of the gammaglutamylcysteine synthetase light subunit by T-butylhydroquinone in HepG2 cells is not dependent on an antioxidant-responsive element. Biochem J 1998;336:535–9. Galloway DC, Blake DG, Shepherd AG, McLellan LI. Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells. Biochem J 1997;328:99–104. Hodnick WF, Duval DL, Pardini RS. Inhibition of mitochondirial respiration and cyanide stimulated generation of reactive oxygen species by selected flavonoids. Biochem Pharmacol 1994;47(3):573– 80. IARC. Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Halogenated acetonitriles. IARC 1991;52:269–86. Ito N, Fukushima S, Hagiwara A, Shibata M, Ogiso T. Carcinogenicity of butylated hydroxyanisole in F344 rats. J Natl Cancer Inst 1983;70:343–52. Jacob S, Abdel-Aziz AA, Shouman SA, Ahmed AE. Effect of glutathione modulation of the distribution and trasnplacental uptake of 2-[14 C]-chloroacetonitrile (CAN) quantitative whole-body autoradiographic study in pregnant mice. Toxicol Ind Health 1998;14(4):533– 46. Jiang J, Xu Y, Klaung JE. Induction of oxidative stress in rat brain by acrylonitrile (CAN). Toxicol Sci 1998;46(2):333–41. Kahl R. Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology 1984;33:185–228. Kondakova LV, Fomina EI, Alekseeva OV. Composition of volatile thermo-destruction components of automobile brake straps. Gig Tr Prof Zabol 1990;3:18–21. Kosower NS, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978;54:109–60. Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium of rat: I. Spermatogenesis and sperm maturation. Ann NY Acad Sci 1952;55:548–73. Li HY, Dai LJ, Quamme GA. Effect of chemical hypoxia on intracellular ATP and cytosolic Mg2+ levels. J Lab Clin Med 1992;122(3):260–72. Li Y, Jaiswal AK. Regulation of human NAD(P)H: quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. J Biol Chem 1992;267:15097–104. Lin EL, Guion CW. Interaction of haloacetonitriles with glutathione and glutathione-S-trasnferase. Biochem Pharmacol 1989;4:685–702. Mashino T, Fridovich I. Mechanism of the cyanide-catalysed oxidation of ␣-ketoaldehydes and ␣-ketoalcohols. Arch Biochem Biophys 1987;252(1):163–70. Matt J. US Patent No. 3,608,684 (1968).
[43] Meier JR, Ringhand HP, Coleman WE, Munch JW, Streicher RP, Kaylor WH, et al. Identification of mutagenic compounds formed during chlorination of humic acid. Mutat Res 1985;157(2–3):111–22. [44] Meister A. Glutatione, ascorbate, and cellular protection. Cancer Res 1994;S4:1960s–75s. [45] Mink DL, Coleman WE, Munch JW, Kaylor WH, Kinghand HP. In vivo formation of halogenated reaction products following peroral sodium hypochlorite. Bull Environ Contam Toxicol 1983;30:394. [46] Moldeus P, Dock L, Cha Y-N, Berggren M, Jernstrom B. Elevation of conjugation capacity in isolated hepatocytes from BHA-treated mice. Biochem Pharmacol 1982;31:1907–10. [47] Monroe D, Eaton D. Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse. Toxicol Appl Pharmacol 1987;90:401–9. [48] Morris RD, Audet AM, Angelillo IF, Chatmers TC, Mosteller F. Chlorination, chlorination-by products and cancer: a meta-analysis. Am J Public Health 1992;82:955–63. [49] Nishizono S, Hayomi T, Ikeda I, Imaizumi K. Protection against the diabetogenic effect by feeding TBHQ to rats prior to the administration of streptozotocin. Biosci Biotechnol Biochem 2000;64:1153–8. [50] Badary OA, Taha RA, Gamal El-Din AM, Abdel-Wahab MH. Thymoquinone is a potent superoxide anion scavenger. Drug Chem Toxicol (in press). [51] Parfitl K. Martindale, the complete drug reference, 32nd ed. London: Pharmaceutical Press; 1999. p. 1126. [52] Prestera T, Zhang Y, Spencer SR, Wilczak CA, Talalay P. The electrophile counter attack response: protection against neoplasia and toxicity. Adv Enzyme Regul 1993;33:281–96. [53] Quast JF, Wade CE, Humiston CG, Carreosn RM, Hermann EA, Park CN, et al. A 2-year toxicity and oncogenicity study with acrylonitrile incorporated into drinking water of rats. Prepared by the toxicology research laboratory, Health and Environmental Research, Dow Chemical USA, Midland, MI; 1980. [54] Rashba-Step J, Turro NJ, Cederbaum AI. Increased NADPH- and NADH-dependent production of superoxide and hydroxyl radicals by microsomes after chronic ethanol treatment. Arch Biochem Biophys 1994;300(1):401–8. [55] Rehwoldt R. Tracking the use of antioxidants through industry surveys. Food Chem Toxicol 1986;24:1039–41. [56] Robatto G, Malinverno G, Bottoman J. Development and implementation of a safety evaluation program for chemical fibers. Regul Toxicol Pharmacol 1993;17:193–208. [57] Rubio R, Galceran MT, Rauret G. Nitriles and isonitriles as interferents in cyanide determination in polluted water. Analysis 1990;115:959–63. [58] Rushmore TH, Pickett CB. Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 1990;265:14648–53. [59] Rushmore TH, Morton MR, Pickett CB. Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 1991;266:11632–9. [60] Saillenfait AM, Bonnet P, Guenier JP, de-Ceaurriz J. Relative developmental toxicities of inhaled aliphatic mononitrile. Fundam Appl Toxicol 1993;20:365–75. [61] Sedlak J, Lindsay RH. Estimation of total protein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192– 205. [62] Solionova LG, Smulevich VB, Turbin EV, Krivosheyeva LV, Plotnikov JV. Carcinogens in rubber production in the Soviet Union. Scand J Work Environ Health 1992;18:120–3. [63] Tandon R, Saxena DK, Chandra SV, Seth PK, Sirvastava SP. Testicular effects of acrylonitrile in mice. Toxicol Lett 1988;42:55– 63.
M.H. Abdel-Wahab / Pharmacological Research 47 (2003) 509–515 [64] Tanii H, Hashimoto K. Influence of ethanol on the in vivo and in vitro metabolism of nitriles in mice. Arch Toxicol 1986;58(3):171–6. [65] Trehy ML, Bieber TL. Detection, identification and quantitative analysis of dihaloacetonitriles in chlorinated natural water. In: Keith LH, editor. Advances in the identification and analysis of organic pollutants in water, vol. 2. Ann Arbor, MI: Ann Arbor Science Publishers; 1981. p. 941–75.
515
[66] Uchiyama M, Mihara M. Determination of malonyldialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86:271–8. [67] Wattenberg LW, Lam LK. In: Nygaard OF, Simig MA, editors. Radioprotectors and anti-carcinogens. New York: Academic Press, Inc.; 1983. p. 461–9.
Testicular toxicity of dibromoacetonitrile and possible protection by tertiary butylhydroquinone Mohamed H. Abdel-Wahab∗ Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt Accepted 21 January 2003
Abstract Dibromoacetonitrile (DBAN) is known to be water disinfectant by-product. Its broad-spectrum toxicity in different test systems in vivo and in vitro has been reported. Oxidative damage induced by DBAN may be partially responsible for its toxicity. Herein, the ability of DBAN to induce oxidative stress in mouse testis and possible protective effect of an antioxidant tertiary butylhydroquinone (TBHQ) were addressed. Male albino mice were injected with a single dose of DBAN (50 mg/kg i.p.), and killed after 3 h of treatment. Control animals received 10 ml/kg body weight i.p. of the vehicle DMSO. In both experiments, cauda epididymis were dissected and sperm count and motility were investigated. Also, testicular activity of lactic dehydrogenase-x (LDH-x) isozyme and histopathological changes were examined. Furthermore, testicular content of malonyldialdehyde (MDA) and reduced glutathione (GSH) were determined. A single i.p. dose of DBAN caused decrease in sperm count and motility to approximately 88 and 84%, respectively, compared with control animals. A 46% decrease in testicular activity of LDH-x, compared with control animals, was observed. A significant accumulation of MDA in DBAN-treated animals was increased to 99% while testicular content of GSH was decreased by 56% compared to control animals. Compared to DBAN-treated animals, treatment with TBHQ (100 mg/kg p.o.) prior exposure to DBAN showed a remarkable degree of protection as indicated by enhancement of sperm count and motility, testicular activity of LDH-x, and GSH. Accumulation of testicular content of MDA significantly decreased following TBHQ treatment compared to DBAN-treated animals. In conclusion, results presented here indicate that DBAN is capable to induce oxidative stress in mouse testis. TBHQ may play a protective role against DBAN-induced testicular cellular damage. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Oxidative stress; Testes; Dibromoacetonitrile; Tertiary butylhydroquinone; Mice
1. Introduction Chlorination of drinking water results in the formation of a variety of hazardous by-products including halogenated acetonitriles (HANs). These by-products are formed as a consequence of the interaction between chlorine and organic impurities in water [30]. Inclusion of bromine in aqueous solutions of chlorine results in preferential substitution of bromine into organic substrates present in water [15]. It has been reported that consumption of chlorinated water could also result in formation of HAN in vivo [45]. HANs have been used as insecticides for stored grains [17], biological growth inhibitors in cooling towers [42] and as common laboratory chemicals [8]. Besides the contaminated drinking water [57], HANs have been detected as environmental hazards, following ingestion of food products which leached by HAN monomers ∗ Tel.:
+20-202-4724450/4724212. E-mail address: [email protected] (M.H. Abdel-Wahab).
from packing materials [16,56], inhalation of cigarette smoke [11], car exhaust [35] and industrial dust in the work place [62,64]. Epidemiological studies have suggested an association between exposure to chlorinated water and the occurrence of gastrointestinal and urinary tract cancers [23,40,48]. Numerous toxicological effects of HAN have been reported. These include mutagenic [20], carcinogenic [14,19,43,53] and embryotoxic [60] effects. Also, testicular adverse effects of HAN and acrylonitrile have been reported [63]. Induction of oxidative damage by these compound and their ability to release cyanide have been associated with the vast majority of reported toxicities [4,32,33]. Dibromoacetonitrile (DBAN, Br2 –CH–CN) is one of the member of HAN group which also exhibits a wide range of toxicity in a manner similar to that described for HAN. DBAN-induced oxidative stress, in different test systems, has been reported by many investigators [3,65]. Although its broad spectrum toxicity in vivo and in vitro has been addressed, its testicular toxicity has not been fully explored.
1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00039-2
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Butylated hydroxyanisole (BHA) and its metabolite tertiary butylhydroquinone (TBHQ) are widely used as food preservatives, probably due to their powerful antioxidant potential [1,2,49,51,55]. It has been reported that, BHA and TBHQ protect animals against radiation and the acute toxicity of various xenobiotics and mutagens [3,4,31,34,67]. Dietary administration of these compounds also leads to the protection against various carcinogens, presumably through the induction of many phase II detoxifying enzymes such as epoxide hydrolases [7,9,12,47], glutathione S-transferases [8,46] and glucuronosyltransferases [18], as well as through the inhibition of cytochrome P-450 monooxygenase [18]. Biochemical and genetic studies show that the induction of these detoxifying enzymes is primarily due to the transcriptional activation of genes and is regulated by an enhancer, called antioxidant-responsive element or electrophile-responsive element [26–28,39,58,59]. Since DBAN has been shown to induce oxidative stress in different test systems; and growing evidences have indicated the association between oxidative stress and male reproductive impairment [5], the present experiment was designed to investigate the potential testicular toxicity of DBAN in male albino mice. Also, the antioxidant potential of TBHQ to modulate the toxicity was examined as an attempt to explain the mechanism of DBAN-induced testicular toxicity.
2. Materials and methods 2.1. Chemicals DBAN was purchased from Aldrich Chemical Co. (Milwaukee, WI). TBHQ was obtained from Aldrich–Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest quality and commercially available. 2.2. Animals Eighteen adult male albino mice, weighing 18–20 g, were obtained from our local animal facility at Al-Azhar University. Mice were maintained in our laboratory under standard laboratory conditions (12 h light:12 h dark, 25 ± 2 ◦ C) with free access to food and water. Animals were divided equally into three groups of six animals each. Control animals received an i.p. injection of DMSO at dose of 10 ml kg−1 once. Six animals received an i.p. injection of DBAN at a dose of 50 mg kg−1 once. The third group of mice received multiple i.p. doses of TBHQ (100 mg kg−1 for 5 consecutive days) prior to exposure to DBAN. 2.3. Methods 2.3.1. Assessment of sperm count and motility Sperm count and motility were determined according to the method of Anderson et al. [6]. Spermatozoa were ob-
tained by making small cuts in the two cauda epididymis from each mouse. The contents of the epididymal spermatozoa were suspended carefully in 20 ml Krebs–Ringer bicarbonate buffer prewarmed to 37 ◦ C. Approximately, 10 l of the suspension was transferred to each chamber of a Neubauer hemocytometer. Over-filling or under-filling was avoided. Separate counts for motile and non-motile sperms in each chamber were taken. If the variation between the chambers exceeds 10%, the whole estimation was repeated. 2.3.2. LDH-x enzyme activity LDH-x activity was assessed according to the method of Cheever et al. [13]. The testes were homogenized in 0.25 M sucrose solution (1:3, w/v) for 1 min. The homogenates were centrifuged at 10,000 × g for 30 min and the supernatant was passed through 0.45-m pore-size acrodisc (Gelman Sciences, Inc., Ann Arbor, MI). The filtrate was used as LDH-x enzyme preparation. Quartz cuvettes containing 3 ml of the specific enzyme substrate were incubated with 20 l of the enzyme preparation for 10 min. The temperature of the reaction mixture was kept at 30 ◦ C and the change in absorbance was measured photometerically at 340 nm. 2.3.3. Determination of reduced glutathione (GSH) Testicular GSH content was determined photometerically following the method of Sedlak and Lindsay [61]. Samples were prepared for non-protein fraction by mixing equal volumes of cooled 10% trichloroacetic acid solution with testicular homogenate. The protein precipitate was removed by centrifugation at 10,000 × g at 4 ◦ C for 10 min. Sulfhydryl concentration was measured, at 412 nm, after addition of 5,5 -dithio-bis (2-nitrobenzoic acid) to the tissue supernatant in the presence of phosphate buffer [22,36]. 2.3.4. Determination of lipid peroxidation Lipid peroxidation was evaluated as malonyldialdehyde (MDA) production by determination of the tissue content of thiobarbituric acid (TBA) reactive substances (TBARSs) as described by Uchiyama and Mihara [66]. Stock solution of 1,1,3,3-tetraethoxypropane in water was used to produce different concentrations of MDA, these solutions were used as TBARS standards. TBA and 1% H3 PO4 were added to homogenates and heated to boiling for 45 min. TBA adducts were extracted with n-butanol. The TBARS concentrations were determined spectrophotometerically utilizing the differences in optical density (OD) at 535 and 520 nm and using the molar absorption constant of 56 × 105 M−1 cm−1 . 2.3.5. Testes histopathology [37,63] For qualitative analysis of testicular histology, one testis from each animal was fixed in Bouin’s fluid for ∼18 h. The fixed testes were embedded in paraffin wax and blocks were prepared. Testicular sections were collected from each testis at 5 m thickness using a rotary microtome and stained with
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haematoxylin and eosin. Histologic examination was performed using light microscopy. Microphotographs were prepared under low and high magnification.
Table 3 Effect of DBAN and TBHQ plus DBAN on the testicular content of GSH and MDA on mice Parameters
2.3.6. Data analysis The GRAPHPAD INPLOT (ISI Software, Philadelphia, PA) computer program was used to plot collected data. Data were expressed as means ± S.E.M. Assessment of the results was performed using one-way ANOVA procedure followed by Bonferroni multiple comparison test using software GRAPHPAD INSTAT (ISI Software). The 0.05 level of probability was used as the criterion for significance.
3. Results The results of sperm count and motility following DBAN and TBHQ plus DBAN are listed in Table 1. I.p. injection of DBAN (50 mg kg−1 , once) caused significant decrease in sperm count and motility by 88 and 84%, respectively, compared with control mice. Treatment of the animals with TBHQ (100 mg kg−1 per day × 5 p.o.) prior to DBAN showed significant decrease of sperm count and motility by 19 and 26%, respectively, compared with DMSO-treated mice. Upon comparing TBHQ-treated mice with DBAN-treated once, TBHQ was shown to increase sperm count and motility by 564 and 372%, respectively. The effect of DBAN and TBHQ plus DBAN on testicular LDH-x isoenzyme activity is given in Table 2. I.p. injection of DBAN caused significant decrease in the
Table 1 Effect of DBAN and TBHQ plus DBAN on sperm count and motility on mice Parameters
Groups Control group
Sperm count (million) % of sperm motility
DBAN-treated group
TBHQ + DBANtreated group
46.3 ± 2.35
5.67 ± 2.36a
37.67 ± 1.33a,b
90.0 ± 1.39
14.17 ± 4.55a
66.83 ± 3.23a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group. Table 2 Effect of DBAN and TBHQ plus DBAN on LDH-x activity on mice Parameter
LDH-x (U/g)
Groups Control group
DBAN-treated group
TBHQ + DBANtreated group
93.4 ± 4.8
50.0 ± 3.6a
115 ± 7.5a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group.
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Groups Control group
GSH (mol/g protein) MDA (nmol/g protein)
1.85 ± 0.05
DBAN-treated group 0.82 ± 0.095a,b
83.00 ± 5.1 165.00 ± 9.32a,b
TBHQ + DBANtreated group 1.6 ± 0.06 115.0 ± 7.5a,b
Results expressed as mean ± S.E.M. of six animals. a Significant from control group. b Significant from DBAN-treated group.
testicular activity of LDH-x by 46% compared with control group. Treatment of mice with TBHQ prior to DBAN caused significant increase in the activity of LDH-x by 23% compared with vehicle-treated animals. Pretreatment of DBAN-treated mice with TBHQ significantly increased the isoenzyme activity by 130% compared with DBAN-treated mice. Concerning the study of the mechanism of testicular toxicity induced by DBAN, the results are shown in Table 3. I.p. injection of DBAN showed a significant decrease in the testicular content of GSH by 56% compared with respective control mice. On the other hand, testicular content of MDA significantly increased by 99% in DBAN-treated animals compared with control. Treatment of the animals with TBHQ prior to DBAN showed significant protective effect as evidenced by normalization of GSH content and mitigation of MDA content. 3.1. Testis histopathology Histopathological examination showed that i.p. injection of DBAN (50 mg kg−1 once) caused marked pathological changes in mice testes (Fig. 1) compared to normal animals (Fig. 3). In the DBAN-treated group, the main damage was found in the seminiferous tubules in the form of oedema patches, pyknosis of spermatogonia and spermocytes, reduction in spermatogonia layers with presence of empty spaces and many bizzar cells. Rupture of basement membrane, swelling and some completely degenerated cells, inhibition of the process of spermatogenesis, presence of giant multinucleated cells and breakdown of the nuclear material (karyolysis) in most cells were observed. Also, degenerated cells were delineated in the seminiferous tubules. Congestion of blood vessels and haemorrhage in the interstitial tissue were noticed. Pretreatment of DBAN-treated animals with TBHQ mitigate some of the previous changes as shown in Fig. 2. This mitigation was in the form of somewhat irregular distribution of the spermatogonic series, basement membrane is slightly affected. Necrotic and karyolysis were less encountered.
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Fig. 1. Pyknosis of spermatids and spermatocytes, oedema, haemorrhage and karyolysis. Magnification: 40×.
Fig. 2. Mitigation of seminiferous tubule of testes. Magnification: 40×.
Fig. 3. Normal seminiferous tubules of testes and presence of spermatogonia. Magnification: 40×.
4. Discussion DBAN, a member of HANs, is produced as a result of chlorination of drinking water. Little information about its potential testicular toxicity as water pollution is available. Hence, this study was designed to investigate the potential testicular toxicity of DBAN and mechanism underlying the
toxicity. Also, possible protective effect of TBHQ, an antioxidant agent, was investigated. The results of the present study showed that single i.p. administration of DBAN in mice significantly decreases the epididymal sperm count and motility compared with control animals. Also DBAN causes histopathological changes in the testis of mice. This is in accordance with a similar
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work of Tandon et al. [63], who reported that exposure of mice to vinyl cyanide (acrylonitrile, VCN) induced damage to the seminiferous epithelium as evidenced by decrease of sperm count and motility. The similarity between our study and the study of Tandon et al. [63] is the release of cyanide ions from both DBAN and VCN metabolism. Cyanide, in turn, interferes with energy production processes required for sperm vitality and motility [24,38]. Also, the testicular activity of LDH-x significantly decreases in DBAN-treated animals. This is in harmony with the study of Abdel Naim [1], who reported that treatment of rats with acrylonitrile inhibited LDH-x activity. This change in LDH-x activity together with the result of sperm count and motility as well as histopathological changes are of valuable index for the testicular toxicity [25]. The mechanism underlying the testicular toxicity of DBAN is not fully explained. However, Ahmed et al. [3] reported that DBAN induced a significant oxidative stress in rat gastric tissues as evidenced by GSH-depletion and GST inhibition. Moreover, induction of oxidative stress was associated with the toxicity of structurally related compounds such as chloroacetonitrile (CAN) [32] and acrylonitrile [4,33]. Our results indicated that i.p. injection of DBAN in mice significantly decreases the testicular content of GSH and increases the content of MDA. This indicates that DBAN induces oxidative stress in testis of mice. This is in the same lines of the study of Jacob et al. [32], Ahmed et al. [4] and Jiang et al. [33]. Several studies have reported the implication of free radicals such as hydroxyl radical and superoxide anion in the testicular toxicity of cyanide-containing compounds such as acrylonitrile [29,41,54]. The formation of reactive oxygen species (OH• , O•− and H2 O2 ) is done through bioactivation of cyanide-containing compounds (VCN, DBAN, CAN) by hepatic microsomal enzymes such as cytochrome-P450 or peroxidases leading to liberation of CN ions which interact with certain compounds inducing free radicals formation [1,2,7,42]. In an attempt to mitigate the testicular toxicity induced by DBAN, we tested the hepatic phase II enzymes induced by TBHQ. The present results showed that, oral administration of TBHQ to the mice prior to DBAN, attenuates the testicular toxicity parameters and mitigates the testicular histopathological changes. This attenuation is due to the potential antioxidant activity of TBHQ [49,52]. Also, the ability of TBHQ to induce hepatic enzymes may play a role [10,44]. TBHQ ameliorates the testicular content of GSH in this study. This may be due to the upregulation of GSH synthesis by TBHQ [21]. The stronger radical scavenger effect of TBHQ may be responsible for the decreased testicular content of MDA [50]. In summary, DBAN has potential testicular toxicity as evidenced by decreased sperm count and motility as well as LDH-x activity. This toxicity was through oxidative stress induction as showed by increased testicular content of MDA and decreased GSH content. The toxicity of DBAN on the testis is mitigated by TBHQ.
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References [1] Abdel Naim AB. Studies on the mechanism of testicular toxicity induced by acrylonitrile. Ph.D. Thesis, Al-Azhar University, Cairo, Egypt; 1995. [2] Ahmed AE, Farooqui MYH, Trieff NM. Nitriles. In: Anders MW, editor. Bioactivation of foreign compounds. Orlando, FL: Academic Press, Inc. p. 485–518. [3] Ahmed AE, Hussein GI, Loh JP, Abdel-Rahman SZ. Studies on the mechanism of haloacetonitrile-induced gastrointestinal toxicity: interaction of dibromoacetonitrile with glutathione and glutathione-S-transferase in rats. J Biochem Toxicol 1991;6(2):115– 21. [4] Ahmed AE, Nouraleen AM, Abdel-Rahman SZ, Rajarman S. Role of glutathione modulation in acrylonitrile-induced gastric DNA damage in rats. Arch Toxicol 1996;70(10):620–7. [5] Ahotupa M, Huhtaniemi I. Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally criptorchid rat testis. Biol Reprod 1992;46:1114–8. [6] Anderson RA, Willis BR, Oswald C, Zaneyeld LJD. Ethanol induced male infertility: impairment of spermatozoa. J Pharmacol Exp Ther 1983;225(2):479–86. [7] Aust SD, Chignell CF, Bxay TM, Kalyanaraman B, Masson RP. Contemporary issues in toxicology. Free radical in toxicology. Toxicol Appl Pharmacol 1993;120:168–78. [8] Barcelo D, Maris FA, Geedink Rb, Frei RW, de Jong GJ, Brinkman UA. Comparison between positive, negative and chloride-enhanced negative chemical ionization of organophosphorus pesticides in on-line liquid chromatography–mass spectrometry. J Chromatogr 1987;8:65–76. [9] Benson AM, Batzinger RP, Ou S-YL, Bueding E, Cha Y-N, Talalay P. Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Cancer Res 1978;38:4486–95. [10] Bock KW, Eckle T, Ouzzine M, Fournel-Gigleux S. Coordinator induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the optical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells. Biochem Pharmacol 2000;59:467–70. [11] Byrd GD, Fowler KW, Hicks RD, Lovette ME, Bovgerding MF. Isotop dilution gas chromatography–mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke. J Chromatogr 1990;503:359–68. [12] Cha YN, Martz F, Beuding E. Enhancement of liver microsome epoxide hydratase activity in rodents by treatment with 2(3)-tert-butyl-4-hydroxyanisole. Cancer Res 1978;38:4496–8. [13] Cheever KL, Weigel WW, Rechards DE, Lal JB, Plotnick HB. Testicular effect of bis (2-methoxyethyl) ether in the adult male rat. Toxicol Ind Health 1989;5(6):1099–109. [14] Clayson DB, Iverson F, Nera EA, Lok E. The significance of induced forestomach tumors. Annu Rev Pharmacol Toxicol 1990;30:441–63. [15] Coleman WE, Munch JW, Kaylor WH, Streicher RP, Ringhand HP, Meier JR. GC/MS analysis of mutagenic extracts of aqueous chlorinated humic acid: comparison of the by-products to drinking water contaminants. Environ Sci Technol 1984;18:674–81. [16] Conacher HB, Page BD, Ryan JJ. Industrial chemical contaminants of foods. Food Addit Contam 1993;10:129–43. [17] Cotton RT, Walkden HH. The role of sorption in the fumigation of stored grain and cereal products. J Kans Entomol Soc 1968;17:98. [18] Cummings SW, Prough RA. Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions. J Biol Chem 1983;258:12315–9. [19] Daniel FB, Schenck KM, Mattox JK, Lin EL, Haas DL, Pereira MA. Genotoxic properties of haloacetonitriles: drinking water by-products of chlorine disinfection. Fundam Appl Toxicol 1986;6(3):447–53. [20] Duverger-van bogaert M, Lambotte-Vandepaer M, De Meester C, Rollmann B, Poncelet F, Mercier M. Effect of several factors on the
514
[21]
[22] [23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36] [37]
[38]
[39]
[40] [41]
[42]
M.H. Abdel-Wahab / Pharmacological Research 47 (2003) 509–515 liver extract mediated mutagenicity of acrylonitrile and identification of four new in vitro metabolites. Toxicol Lett 1981;7:311–9. Eftekhar EP, Arne H, Bernhord HJ. Thioredoxin reductase and glutathione synthesis is regulated by TBHQ in cortical astrocytes but not in cortical neurons. GLIA 2000;31:241–8. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;74:214–26. Flaten TP. Chlorination of drinking water and cancer incidence in Norway. Int J Epidemiol 1992;21:6–15. Folgero T, bertheussen K, Lindal S, Toybergsen T, Oian P. Mitochondirial disease and reduced sperm motility. Hum Reprod 1993;8(11):1863–8. Foster PMD, Blackburn DM, Moore RB, Lloyd SC. Testicular toxicity of 2-methoxyacetaldehyde, a possible metabolite of ethylene glycol monomethyl ether in the rat. Toxicol Lett 1986;32:289–314. Friling RS, Bensimon A, Tichauer Y, Daniel V. Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proc Natl Acad Sci USA 1990;87:6258–62. Galloway DC, McLellan LT. Inducible expression of the gammaglutamylcysteine synthetase light subunit by T-butylhydroquinone in HepG2 cells is not dependent on an antioxidant-responsive element. Biochem J 1998;336:535–9. Galloway DC, Blake DG, Shepherd AG, McLellan LI. Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells. Biochem J 1997;328:99–104. Hodnick WF, Duval DL, Pardini RS. Inhibition of mitochondirial respiration and cyanide stimulated generation of reactive oxygen species by selected flavonoids. Biochem Pharmacol 1994;47(3):573– 80. IARC. Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Halogenated acetonitriles. IARC 1991;52:269–86. Ito N, Fukushima S, Hagiwara A, Shibata M, Ogiso T. Carcinogenicity of butylated hydroxyanisole in F344 rats. J Natl Cancer Inst 1983;70:343–52. Jacob S, Abdel-Aziz AA, Shouman SA, Ahmed AE. Effect of glutathione modulation of the distribution and trasnplacental uptake of 2-[14 C]-chloroacetonitrile (CAN) quantitative whole-body autoradiographic study in pregnant mice. Toxicol Ind Health 1998;14(4):533– 46. Jiang J, Xu Y, Klaung JE. Induction of oxidative stress in rat brain by acrylonitrile (CAN). Toxicol Sci 1998;46(2):333–41. Kahl R. Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology 1984;33:185–228. Kondakova LV, Fomina EI, Alekseeva OV. Composition of volatile thermo-destruction components of automobile brake straps. Gig Tr Prof Zabol 1990;3:18–21. Kosower NS, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978;54:109–60. Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium of rat: I. Spermatogenesis and sperm maturation. Ann NY Acad Sci 1952;55:548–73. Li HY, Dai LJ, Quamme GA. Effect of chemical hypoxia on intracellular ATP and cytosolic Mg2+ levels. J Lab Clin Med 1992;122(3):260–72. Li Y, Jaiswal AK. Regulation of human NAD(P)H: quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. J Biol Chem 1992;267:15097–104. Lin EL, Guion CW. Interaction of haloacetonitriles with glutathione and glutathione-S-trasnferase. Biochem Pharmacol 1989;4:685–702. Mashino T, Fridovich I. Mechanism of the cyanide-catalysed oxidation of ␣-ketoaldehydes and ␣-ketoalcohols. Arch Biochem Biophys 1987;252(1):163–70. Matt J. US Patent No. 3,608,684 (1968).
[43] Meier JR, Ringhand HP, Coleman WE, Munch JW, Streicher RP, Kaylor WH, et al. Identification of mutagenic compounds formed during chlorination of humic acid. Mutat Res 1985;157(2–3):111–22. [44] Meister A. Glutatione, ascorbate, and cellular protection. Cancer Res 1994;S4:1960s–75s. [45] Mink DL, Coleman WE, Munch JW, Kaylor WH, Kinghand HP. In vivo formation of halogenated reaction products following peroral sodium hypochlorite. Bull Environ Contam Toxicol 1983;30:394. [46] Moldeus P, Dock L, Cha Y-N, Berggren M, Jernstrom B. Elevation of conjugation capacity in isolated hepatocytes from BHA-treated mice. Biochem Pharmacol 1982;31:1907–10. [47] Monroe D, Eaton D. Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse. Toxicol Appl Pharmacol 1987;90:401–9. [48] Morris RD, Audet AM, Angelillo IF, Chatmers TC, Mosteller F. Chlorination, chlorination-by products and cancer: a meta-analysis. Am J Public Health 1992;82:955–63. [49] Nishizono S, Hayomi T, Ikeda I, Imaizumi K. Protection against the diabetogenic effect by feeding TBHQ to rats prior to the administration of streptozotocin. Biosci Biotechnol Biochem 2000;64:1153–8. [50] Badary OA, Taha RA, Gamal El-Din AM, Abdel-Wahab MH. Thymoquinone is a potent superoxide anion scavenger. Drug Chem Toxicol (in press). [51] Parfitl K. Martindale, the complete drug reference, 32nd ed. London: Pharmaceutical Press; 1999. p. 1126. [52] Prestera T, Zhang Y, Spencer SR, Wilczak CA, Talalay P. The electrophile counter attack response: protection against neoplasia and toxicity. Adv Enzyme Regul 1993;33:281–96. [53] Quast JF, Wade CE, Humiston CG, Carreosn RM, Hermann EA, Park CN, et al. A 2-year toxicity and oncogenicity study with acrylonitrile incorporated into drinking water of rats. Prepared by the toxicology research laboratory, Health and Environmental Research, Dow Chemical USA, Midland, MI; 1980. [54] Rashba-Step J, Turro NJ, Cederbaum AI. Increased NADPH- and NADH-dependent production of superoxide and hydroxyl radicals by microsomes after chronic ethanol treatment. Arch Biochem Biophys 1994;300(1):401–8. [55] Rehwoldt R. Tracking the use of antioxidants through industry surveys. Food Chem Toxicol 1986;24:1039–41. [56] Robatto G, Malinverno G, Bottoman J. Development and implementation of a safety evaluation program for chemical fibers. Regul Toxicol Pharmacol 1993;17:193–208. [57] Rubio R, Galceran MT, Rauret G. Nitriles and isonitriles as interferents in cyanide determination in polluted water. Analysis 1990;115:959–63. [58] Rushmore TH, Pickett CB. Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 1990;265:14648–53. [59] Rushmore TH, Morton MR, Pickett CB. Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 1991;266:11632–9. [60] Saillenfait AM, Bonnet P, Guenier JP, de-Ceaurriz J. Relative developmental toxicities of inhaled aliphatic mononitrile. Fundam Appl Toxicol 1993;20:365–75. [61] Sedlak J, Lindsay RH. Estimation of total protein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192– 205. [62] Solionova LG, Smulevich VB, Turbin EV, Krivosheyeva LV, Plotnikov JV. Carcinogens in rubber production in the Soviet Union. Scand J Work Environ Health 1992;18:120–3. [63] Tandon R, Saxena DK, Chandra SV, Seth PK, Sirvastava SP. Testicular effects of acrylonitrile in mice. Toxicol Lett 1988;42:55– 63.
M.H. Abdel-Wahab / Pharmacological Research 47 (2003) 509–515 [64] Tanii H, Hashimoto K. Influence of ethanol on the in vivo and in vitro metabolism of nitriles in mice. Arch Toxicol 1986;58(3):171–6. [65] Trehy ML, Bieber TL. Detection, identification and quantitative analysis of dihaloacetonitriles in chlorinated natural water. In: Keith LH, editor. Advances in the identification and analysis of organic pollutants in water, vol. 2. Ann Arbor, MI: Ann Arbor Science Publishers; 1981. p. 941–75.
515
[66] Uchiyama M, Mihara M. Determination of malonyldialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86:271–8. [67] Wattenberg LW, Lam LK. In: Nygaard OF, Simig MA, editors. Radioprotectors and anti-carcinogens. New York: Academic Press, Inc.; 1983. p. 461–9.