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Induction of CYP2E1 in Testes of Isoniazid-treated Rats as Possible Cause of Testicular Disorders.
... Even though the most commonly reported normal tissue toxicity is hepatic toxicity (Jaswal et al. 2013;Adaramoye et al. 2016;Zhang et al. 2016), there are reports about optic toxicity (Ayanniyi and Ayanniyi 2011), nephrotoxicity (Chang et al. 2014), neurotoxicity (Kass and Shandera 2010), and reproductive toxicity (Awodele et al. 2013;Ahmadi et al. 2016;Rao et al. 2020). Preclinical studies have demonstrated the male reproductive tissue toxicity of ATDs when administered individually (Bondarenko et al. 2012;Shayakhmetova et al. 2015;Rao et al. 2016Rao et al. , 2020 Rao et al. 2016), elevated DNA damage (Shayakhmetova et al. 2015(Shayakhmetova et al. , 2017, oxidative stress (Shayakhmetova et al. 2015) and poor fertilising ability of spermatozoa (Bondarenko et al. 2011(Bondarenko et al. , 2012 are common consequences of ATDs on male reproductive function. On the contrary, there is limited information on the effects of ATDs on the female reproductive system. ...
... Even though the most commonly reported normal tissue toxicity is hepatic toxicity (Jaswal et al. 2013;Adaramoye et al. 2016;Zhang et al. 2016), there are reports about optic toxicity (Ayanniyi and Ayanniyi 2011), nephrotoxicity (Chang et al. 2014), neurotoxicity (Kass and Shandera 2010), and reproductive toxicity (Awodele et al. 2013;Ahmadi et al. 2016;Rao et al. 2020). Preclinical studies have demonstrated the male reproductive tissue toxicity of ATDs when administered individually (Bondarenko et al. 2012;Shayakhmetova et al. 2015;Rao et al. 2016Rao et al. , 2020 Rao et al. 2016), elevated DNA damage (Shayakhmetova et al. 2015(Shayakhmetova et al. , 2017, oxidative stress (Shayakhmetova et al. 2015) and poor fertilising ability of spermatozoa (Bondarenko et al. 2011(Bondarenko et al. , 2012 are common consequences of ATDs on male reproductive function. On the contrary, there is limited information on the effects of ATDs on the female reproductive system. ...
... Even though the most commonly reported normal tissue toxicity is hepatic toxicity (Jaswal et al. 2013;Adaramoye et al. 2016;Zhang et al. 2016), there are reports about optic toxicity (Ayanniyi and Ayanniyi 2011), nephrotoxicity (Chang et al. 2014), neurotoxicity (Kass and Shandera 2010), and reproductive toxicity (Awodele et al. 2013;Ahmadi et al. 2016;Rao et al. 2020). Preclinical studies have demonstrated the male reproductive tissue toxicity of ATDs when administered individually (Bondarenko et al. 2012;Shayakhmetova et al. 2015;Rao et al. 2016Rao et al. , 2020 Rao et al. 2016), elevated DNA damage (Shayakhmetova et al. 2015(Shayakhmetova et al. , 2017, oxidative stress (Shayakhmetova et al. 2015) and poor fertilising ability of spermatozoa (Bondarenko et al. 2011(Bondarenko et al. , 2012 are common consequences of ATDs on male reproductive function. On the contrary, there is limited information on the effects of ATDs on the female reproductive system. ...
Context:
Tuberculosis is one of the major infectious diseases, with people of reproductive age group having a high risk of infection.
Aims:
The present study was designed to understand the consequences of anti-tuberculosis drugs (ATDs) used in DOTS (directly observed treatment short course) schedule on ovarian function.
Methods:
Adult female Swiss albino mice were orally administered with combinations of ATDs used in the DOTS schedule every day for 4weeks. At 2weeks after the cessation of ATDs administration, the endocrine changes and ovarian function were assessed in mice.
Key results:
Administration of ATDs to mice resulted in a prolonged estrous cycle, reduced ovarian follicle reserve, alteration in FSH, LH, and progesterone level, and decreased the number of ovulated oocytes. Further, the degree of fragmentation, degeneration, abnormal distribution of cytoplasmic organelles, abnormal spindle organisation, and chromosomal misalignment were higher in oocytes that were ovulated following superovulation. Blastocysts derived from ATDs treated mice had significantly lower total cell numbers and greater DNA damage. A marginal increase in the number of resorbed fetuses was observed in all the ATDs treated groups except in the multidrug resistance treatment group. Male progeny of ATDs treated mice had decreased sperm count and lower progressive motility, while female progeny exhibited a non-significant reduction in the number of oocytes ovulated.
Conclusions:
Theresults of this study suggest that ATDs can have significant adverse effects on the ovarian reserve, cytoplasmic organisation of oocytes, and can potentially cause transgenerational changes.
Implications:
The findings of the present study indicate ovarian toxicity of ATDs and warrant further research in the direction of identifying alternate drugs with minimal toxicity, and strategies to mitigate the ovarian toxicity induced by these drugs.
... Alp et al. [25] demonstrated that isoniazid and streptomycin induced testicular damage, lowered epididymal semen quality, and reduced gonadotropins and testosterone. Shayakhmetova et al. [26] demonstrated that isoniazid significantly reduced circulatory testosterone, sperm count, fertility index by promoting testicular lipid peroxidation and DNA fragmentation through induction of cytochrome P-450 2E1 (CYP2E1) in Wistar rats. In another study, Shayakhmetova and his colleagues also observed that ethambutol exerted a similar effect as isoniazid [27] in Wistar rats. ...
... In contrast, the HAART + Anti-Koch-treated animals received co-treatment of HAART and anti-Koch as anti-Koch-treated and HAART-treated rats. Human Equivalent Doses of the drugs for rats (HAART: 52.9 mg/kg of Efavirenz, 26 [21,23]. ...
... Spermatogenesis was evaluated by histological scoring of total and daily sperm production [35], mean testicular biopsy score using Johnson's scoring [34], germ cell layer [36,37], and spermatogenic index using four points system [26]. In assessing total and daily sperm production, the tunica albuginea was removed from the testes, and the parenchyma was homogenized in 5 mL of saline-Triton 0.5 % for 30 s. ...
Highly active anti-retroviral therapy (HAART) is an effective anti-retroviral cocktail. Similarly, anti-Koch is highly potent against Mycobacterium tuberculosis. However, these drugs have been shown to impair male fertility. This study investigated the impact of HAART and anti-Koch, when used alone and co-administered, on testicular and sperm integrity. Thirty-two adult male Wistar rats were assigned randomly into four groups (n = 8), namely normal control, HAART-treated, anti-Koch-treated, and HAART + anti-Koch-treated. The doses of drugs were the human equivalent doses for rats. Administration was once daily per os and lasted for eight weeks. HAART aggravated anti-Koch-induced reduction in testicular and penile weights. In addition, anti-Koch also led to a distortion of testicular cytoarchitecture, disturbed spermatogenesis, and caused low sperm quality, including sperm dysmotility. More so, anti-Koch led to a significant elevation of uric acid and dysregulation of testicular lactate transport and glutathione content. These events were accompanied by enhanced lipid peroxidation and inflammation of the testicular tissue and reduced testicular and sperm DNA integrity. These adverse effects of anti-Koch were aggravated by co-administration of HAART. Thus, our results infer that HAART exacerbates anti-Koch-induced impairment of spermatogenesis and testicular and sperm toxicity through up-regulation of uric acid generation and dysregulation of lactate transport and glutathione system.
... We have previously shown the anti-fertility effect of coadministered ATD in male rats [8,9]. It has been reported that the co-administration of ethambutol (EMB), isoniazid (INH), rifampin (RMP) and pyrazinamide (PZA) (in therapeutic doses) to male rats during the period of spermatogenesis caused a modulation Central ...
... Previously we have shown increase in CYP2Е1 mRNA content in rats testes following EMB, INH, RMP and PZA co-administration [8], as well as increase in CYP2Е1 mRNA content and enzymatic activity in liver [36]. Moreover, it has been reported that INH induces CYP2Е1 in the liver and testes [9,[27][28][29]. Thus, we speculate that a more pronounced increase in the expression of CYP2E1 gene with alcohol and ATD co-administration could be due to synergistic or additive effects of different factors at the level of transcription. ...
... Anyway, increased expression of CYP2E1 in the rats' testes of both experimental groups could have serious toxicological outcomes due to CYP2Е1 ability for massive generation of oxygen reactive species (ROS), such as superoxide radical [23]. Increased oxidative stress at the level of testicles after repeated administration of alcohol or ADT has been confirmed by our previous findings and those of other authors [7,9,[35][36][37][38][39][40][41][42][43][44]. Oxidative stress can lead to serious disruption of nucleic acids and proteins metabolism, structure and functions and may play an important role in induced DNA disruption in the germ cells [45]. ...
Alcohol is well documented to affect male fertility. The association between alcohol use disorders and tuberculosis has long been known and well described. Taking into account that both alcohol and anti-tuberculosis drugs (ATD) could have negative impacts on male gonads, an increase in adverse effects is probable. The study aimed to determine combine effects of long-term alcohol consumption and ATD administration on male rat testes CYP2E1 mRNA and protein expression, DNA fragmentation, spermatogenesis parameters, as well as on male reproductive capacity and antenatal development of its posterity. Wistar albino male rats were divided into three groups: I – control (intact animals), II – chronic alcoholism (15% ethanol self-administration during 150 days): III-chronic alcoholism + ATD (ethambutol, isoniazid, rifampin and pyrazinamide) administration. ombined effects of chronic alcoholism and ATD led to changes in testicular CYP2E1 mRNA and protein expression, as well as in DNA fragmentation processes. These abnormalities were more expressed in alcoholic with ATD co-administration group as compared with alcoholic group. The revealed disorders at levels of testicular genome and proteome caused negative implications on spermatogenic epithelium quantitative and qualitative parameters, sperm count, male fertility, and postimplantational surviving of its offspring. Thus the ATD administration modulated the impact of long-term alcohol consumption on spermatogenesis and aggravated paternal-mediated negative effects on posterity.
... On the other hand, studies have reported that the administration of anti-Koch's cocktail containing ethambutol, isoniazid, rifampicin and pyrazinamide triggers oxidative testicular and sperm damage (Shayakhmetova et al., 2012). Anti-Koch's-induced reproductive toxicity has been linked with induction of testicular cytochrome P-450 2EI mRNA expression which triggers generation of reactive oxygen species and other toxic metabolites that mediates DNA damage, impaired spermatogenesis and reduced male fertility (Shayakhmetova et al., 2015(Shayakhmetova et al., , 2017. Although the efficacy of anti-Koch's therapy against Mycobacterium tuberculosis is undoubted, their side effects, particularly reproductive toxicity, have compromised its adherence. ...
... These findings were associated with a reduced survival in offspring whose 'fathers' were treated with HAART, anti-Koch's and HAART + Anti-Koch's. These observations are in tandem with previous studies that documented reduced fertility capacity, lower litter size and weight and reduced survival of offspring of HAART and/or anti-Koch's-treated parents (Onwuamah et al., 2014;Shayakhmetova et al., 2015Shayakhmetova et al., , 2017). The reduced fertility success and fertility index are, at least partly, secondary to HAART-and Anti-Koch's-caused impaired sexual performance and poor sperm quality, while the observed lower litter size and weight might be due to an epigenetic modification with alterations in offspring quality. ...
This study investigated the impact of the administration of HAART and anti‐Koch's, singly and in combination, on sexual competence and birth statistics. Adult male Wistar rats were randomised into distilled water‐treated control, HAART‐treated, anti‐Koch's‐treated and HAART + anti‐Koch's‐treated groups. The 56‐day oral treatment led to impaired sexual competence evident by significantly reduced motivation to mate, prolonged latencies of mount, intromissions, ejaculations and post‐ejaculatory interval, as well as reduced frequencies of mount, intromissions and ejaculations. This was accompanied by significant reductions in penile erection reflex and penile grooming. HAART and anti‐Koch's, when administered singly or in combination, also led to significant reductions in the circulatory follicle‐stimulating hormone, luteinizing hormone, testosterone and intratesticular testosterone, but a significant rise in prolactin. Also, HAART and/or anti‐Koch's significantly reduced sperm count, sperm motility, sperm viability and spermatozoa with normal morphology. Furthermore, HAART and anti‐Koch's, separately or in combination, significantly lowered fertility capacity, litter size and litter weight and offspring survival. The deleterious effects of these drugs were more pronounced when combined. Findings of the present study revealed that HAART and/or anti‐Koch's impair sexual competence via a testosterone‐dependent hyperprolactinemia‐mediated mechanism. These events are associated with reduced fertility capacity, poor sperm quality and lowered offspring survival.
... Positive links between INH, inflammation by endotoxic LPS, and CYP2E1 already have been established, as both INH and LPS provoked CYP2E1 expression, which in return increases the sensitivity of body organs, including the liver, to their potential toxicities (Yue et al., 2004;Cheng et al., 2013;Shayakhmetova et al., 2015). Moreover, CYP2E1-induced oxidative stress and ROS production have been directly associated with liver damage (Rendic, 1999;Ramaiah et al., 2001;Abdelmegeed et al., 2012). ...
... Documentations regarding participations of both INH and its metabolites in the induction of hepatic CYP2E1 are available (Yue et al., 2004;Cheng et al., 2013). Meanwhile, reports regarding the link between LPS and CYP2E1 revealed that both LPS and CYP2E1 were found to be sharing a unique relationship in which bacterial LPS not only stimulates CYP2E1 expression, but also increases the hepatic sensitivity toward CYP2E1 that further exaggerates INH/LPS toxicity (Lu and Cederbaum, 2010;Cederbaum et al., 2012;Shayakhmetova et al., 2015). DAS has attracted a particular interest as a potential therapeutic or prophylactic agent because of its inhibitory actions on CYP2E1-mediated metabolic activation of various chemicals and carcinogens. ...
Tuberculosis (TB) is one of the oldest infectious diseases that affected humankind and remains one of the world’s deadliest communicable diseases that could be considered as global emergency, but the discovery and development of isoniazid (INH) in the 1950s paved the way to an effective single and/or combined first-line anti-TB therapy. However, administration of INH induces severe hepatic toxicity in some patients. Previously, we establish a rat model of INH hepatotoxicity utilizing the inflammatory stress theory, in which bacterial lipopolysaccharide (LPS) potentially enhanced INH toxicity. These enhancing activities ranged between augmenting the inflammatory stress, oxidative stress, alteration of bile acid homeostasis, and CYP2E1 over-expression. Although pre-treatment with dexamethasone (DEX) helped overcome both inflammatory and oxidative stress which ended-up in alleviation of LPS augmenting effects, but still minor toxicities were being detected, alongside with CYP2E1 over expression. This finding positively indicated the corner-stone role played by CYP2E1 in the pathogenesis of INH/LPS-induced liver damage. Therefore, we examined whether INH/LPS co-treatment with CYP2E1 inhibitor diallyl sulfide (DAS) and DEX can protect against the INH/LPS-induced hepatotoxicity. Our results showed that pre-administration of both DAS and DEX caused significant reduction in serum TBA, TBil, and gamma-glutamyl transferase levels. Furthermore, the histopathological analysis showed that DAS and DEX could effectively reverse the liver lesions seen following INH/LPS treatment and protect against hepatic steatosis as indicated by absence of lipid accumulation. Pre-treatment with DAS alone could not completely block the CYP2E1 protein expression following INH/LPS treatment, as appeared in the immunoblotting and immunohistochemistry results. This is probably due to the fact that the combined enhancement activities of both INH and LPS on CYP2E1 protein expression levels might resist the blocking probabilities of DAS. In the meantime, addition of DEX to the DAS/INH/LPS combination caused a significant reduction in CYP2E1 protein expression as revealed by the immunoblotting and fading coloration in immunohistochemistry results. Thus, addition of DEX and DAS together caused strong protection against INH/LPS-induced hepatic damage. These findings reveal the potential therapeutic value of combining DAS and DEX with INH in TB management for reducing the potential risk and incidences of hepatotoxicity.
... Many researchers, including our previous report, focused on the core role played by CYP2E1 in INH toxicity (Yue et al., 2004;Cheng et al., 2013;Shayakhmetova et al., 2015;Hassan et al., 2016), we found a positive correlation between INH hepatotoxicity and CYP2E1. Spectacularly, CYP2E1 at its gene expression level almost dwindled in all INH/LPStreated rats, even those already receiving DEX ( Figure 5E). ...
... INH and its metabolites are responsible for hepatic CYP2E1 induction (Yue et al., 2004;Cheng et al., 2013). Meanwhile, LPS and CYP2E1 shared a unique relationship in which bacterial LPS not only stimulating CYP2E1 expression, but also increases the hepatic sensitivity toward CYP2E1 that further exaggerates INH/LPS toxicity (Lu and Cederbaum, 2010;Cederbaum et al., 2012;Shayakhmetova et al., 2015). Our results clearly indicated that DEX assisted in marginal reduction of CYP2E1 expression, but its capability to fully block CYP2E1 was unsuccessful. ...
Isoniazid (INH) remains a cornerstone key constitute of the current tuberculosis management strategy, but its hepatotoxic potentiality remains a significant clinical problem. Our previous findings succeed to establish a rat model of INH hepatotoxicity employing the inflammatory stress theory in which non-injurious doses of inflammatory-mediating agent bacterial lipopolysaccharides (LPS) augmented the toxicity of INH that assist to uncover the mechanisms behind INH hepatotoxicity. Following LPS exposure, several inflammatory cells are activated and it is likely that the consequences of this activation rather than direct hepatocellular effects of LPS underlie the ability of LPS to augment toxic responses. In this study, we investigated the potential protective role of the anti-inflammatory agent dexamethasone (DEX), a potent synthetic glucocorticoid, in INH/LPS hepatotoxic rat model. DEX pre-treatment successfully eliminates the components of the inflammatory stress as shown through analysis of blood biochemistry and liver histopathology. DEX potentiated hepatic anti-oxidant mechanisms while serum and hepatic lipid profiles were reduced. However, DEX administration was not able to revoke the principal effects of cytochrome P450 2E1 (CYP2E1) in INH/LPS-induced liver damage. In conclusion, this study illustrated the DEX-preventive capabilities on INH/LPS-induced hepatotoxicity model through DEX-induced potent anti-inflammatory activity whereas the partial toxicity seen in the model could be attributed to the expression of hepatic CYP2E1. These findings potentiate the clinical applications of DEX co-administration with INH therapy in order to reduce the potential incidences of hepatotoxicity.
... 3 an increase in the rate of thiobarbituric acid-reactive substances (TBARS) formation in rats testes and sperm, decrease in testis glutathione and protein SH-group contents, significant changes in DNA fragmentation, fatal reduction of male fertilizing capacity and fertility, and an increase in pre-and postimplantation embryo-lethality, increased content of testicular cytochrome P-450 isoenzymes (CYP2E1, CYP3A2, and CYP2C23) messenger RNA (mRNA) with simultaneous morphological and morphometric changes in spermatogenic epithelium. [4][5][6] Taking into account our previous results referred to alterations of testicular CYP2E1 following INH and antitubercular drugs combination administration, it is important to note that this particular isozyme is localized in Leydig cells, where testosterone (TS) biosynthesis occurs. 7,8 Consequently, these structures, and their microenvironment damage as a result of CYP2E1-mediated processes presumably could be the causes of steroidogenesis inhibition and spermatogenesis disruption. ...
... [34][35][36] In addition, our recent experiments demonstrated induction of testicular CYP2E1 following INH administration. 6 Important to note, that interactions between other anti-tuberculosis drugs and CYP2E1 have not been investigated yet extensively. We have demonstrated the significant increase of CYP2E1 mRNA and protein levels in testes, as well as marked activation of CYP2E1-catalyzed oxidation of PNP. ...
Ethambutol (EMB) is conventionally used to treat tuberculosis and atypical Mycobacterium infections in combination with other antimycobacterial drugs. Eventually, EMB testicular toxicity has not been explored extensively yet. The aim of the study is to evaluate testicular toxicity of EMB. We explored the impact of EMB on male rats’ fertility, testosterone level and germ cells state, testicular pro- and anti-oxidant status and DNA damage, as well as identified EMB effects on cytochrome P-450 2E1 (CYP2E1) both with computer simulation and in vivo. We demonstrated that EMB administration to male rats decreased in epididymal sperm count (19%) and fertility index (53%). These events were accompanied by reduction in serum testosterone content (1.6 times) and appearance of spermatogenic epithelium damages. It was also found in testes the intensification of lipid peroxidation, decrease in reduced glutathione content and changes in DNA fragmentation. Additionally, computer simulation showed direct interaction of EMB with CYP2E1 active site and heme. On the top of this, we demonstrated that level of testicular CYP2E1 messenger RNA in EMB-treated rats was increased 8.7 folds and p-nitrophenol hydroxylase activity in testes rose three folds. As this shows, EMB-caused CYP2E1 induction, oxidative stress, and apoptosis in the testes contribute to inhibition of steroidogenesis enzymes and spermatogenesis disruption.
... There are several studies in the literatures suggesting that ATDs have the potential to cause liver injury leading to hepatitis [2,5,[7][8]. Other than hepatotoxicity, ATDs also results in nephrotoxicity [9], neurotoxicity [10], phototoxicity [11], ocular toxicity [12][13] reproductive toxicity [14][15]. ...
This study was aimed to observe the multiple organ toxicities induced by different anti-tubercular drug resistant groups like isoniazid resistance, rifampicin resistance, multidrug resistance including drug susceptible anti-tubercular drugs and their time of recovery from the toxicities induced by these drugs. Wistar Albino mice were treated with drug susceptible anti-tubercular drug (Isoniazid, Rifampicin, Ethambutol & Pyrazinamide), Isoniazid resistance anti-tubercular drugs (Kanamycin, Levofloxacin, Rifampicin, Pyrazinamide & Ethambutol), Rifampicin resistance anti-tubercular drugs (Kanamycin, Levofloxacin, Ethionamide, Cycloserine, Isoniazid, Pyrazinamide & Ethambutol), Multidrug resistance anti-tubercular drug (Kanamycin, Levofloxacin, Ethionamide, Cycloserine, Pyrazinamide & Ethambutol). Animals were sacrificed after giving two weeks recovery gap from the last drug treatment. To understand the late effects, mice were sacrificed after giving two months recovery gap from the last drug treatment. Histopathology of internal organs was done by H & E and PAS. Bone marrow cells were stained with Acridine Orange dye to detect micronucleus and apoptotic cells. Expression of γ-H2AX in bone marrow cells were done by immunohistochemistry. In liver moderate to severe type of toxicities were observed in all groups in the form of nuclear alteration, sinusoidal dilation, vacuolization, necrosis, WBC infiltration. In kidney we had observed mesangial cell proliferation, vacuolization, WBC infiltration, dilation of Bowman’s space. The severity of toxicity was more at short term recovery group than long term recovery groups. Drug susceptible anti-tubercular drugs (INH, RIF, and EMB & PZA) had shown more toxic in compared to other anti-tubercular drugs.
... However, some lumen appeared fibrotic with maturation arrest and presence of some degenerated germ cells (white arrow). Spermatogenic index was determined using the four points system (Shayakhmetova et al., 2015). The number of cell layers, cell types, and the presence of late spermatids in the seminiferous tubules were scored as follows: ...
Andrographis paniculata has been shown to be associated with male reproductive dysfunction, although the available data are scarce and inconsistent, and the associated mechanisms are elusive. Hormonal mechanism via hypothalamic-pituitary-testicular axis, and non-hormonal mechanism primarily through oxidative stress, are involved in the modulation of male reproductive function. We therefore, hypothesized that suppression of hypothalamic-pituitary-testicular axis and/or oxidative stress is involved in Andrographis paniculata-induced reproductive dysfunction. Male Wistar rats received either vehicle or Andrographis paniculata in varying doses of 250, 500, and 1000 mg/kg body weight daily for 8 weeks. Treatment with Andrographis paniculata led to reduced sperm count, motility, and viability. Andrographis paniculata treatment also resulted in distorted spermatogenesis and reduced serum testosterone. On the other hand, Andrographis paniculata led to reduction in the testicular content of malondialdehyde, nitric oxide, TNF-α, and IL-6, and testicular activities of xanthine oxidase and myeloperoxidase, but raised testicular levels of reduced glutathione content and enhanced activity of super oxide dismutase. However, body weight gain, and absolute and relative reproductive organ weights were similar across all the groups. These findings demonstrate that Andrographis paniculata induces reproductive toxicity via suppression of testosterone and not induction of oxidative stress. Therefore, Andrographis paniculata could be a potential and safe male contraceptive.
... Similar changes were observed in rats treated with isoniazid. Isoniazid led to testicular damage with germ cell malformation, including the formation of large multinucleate germ cells (144). ...
Vitamin B-6 in the form of pyridoxine (PN) is commonly used by the general population. The use of PN-containing supplements has gained lots of attention over the past years as they have been related to the development of peripheral neuropathy. In light of this, the number of reported cases of adverse health effects due to the use of vitamin B-6 have increased. Despite a long history of study, the pathogenic mechanisms associated with PN toxicity remain elusive. Therefore, the present review is focused on investigating the mechanistic link between PN supplementation and sensory peripheral neuropathy. Excessive PN intake induces neuropathy through the preferential injury of sensory neurons. Recent reports on hereditary neuropathy due to pyridoxal kinase (PDXK) mutations may provide some insight into the mechanism, as genetic deficiencies in PDXK lead to the development of axonal sensory neuropathy. High circulating concentrations of PN may lead to a similar condition via the inhibition of PDXK. The mechanism behind PDXK-induced neuropathy is unknown; however, there is reason to believe that it may be related to γ-aminobutyric acid (GABA) neurotransmission. Compounds that inhibit PDXK lead to convulsions and reductions in GABA biosynthesis. The absence of central nervous system-related symptoms in PDXK deficiency could be due to differences in the regulation of PDXK, where PDXK activity is preserved in the brain but not in peripheral tissues. As PN is relatively impermeable to the blood–brain barrier, PDXK inhibition would similarly be confined to the peripheries and, as a result, GABA signaling may be perturbed within peripheral tissues, such as sensory neurons. Perturbed GABA signaling within sensory neurons may lead to excitotoxicity, neurodegeneration, and ultimately, the development of peripheral neuropathy. For several reasons, we conclude that PDXK inhibition and consequently disrupted GABA neurotransmission is the most plausible mechanism of toxicity.
... However, studies on assessing the effect of these drugs on reproductive system are very few. Pyrazinamide (Bondarenko et al., 2011, streptomycin (Alp et al., 2012), isoniazid (Shayakhmetova et al., 2015) and ethambutol (Rao et al., 2016;Shayakhmetova et al., 2016) are reported to exert testicular toxicity. Co-administration of EMB, RIF, INH and PZA has shown to reduce the testicular glutathione (GSH) level and increase in pre and post-implantation embryo lethality in rats . ...
... Therefore, we speculated that flavonoids might be active compounds that can reduce the impact of liverdamaging substances by inhibiting the expression of CYP2E1, which might be one of the hepatoprotective mechanisms of flavonoids. Also, an investigation of the effects of flavonoids on CYP2E1 expression is necessary since some daily foods (purple potato, purple sweet potato, broccoli, bitter gourd, garlic, and tomato), beverages (coffee and cacao polyphenol), and drugs (Schisandra chinensis, danshen, Gelsemium elegans, isoniazid, diallyl sulfide, sulforaphane, and tamoxifen) are metabolized by CYP2E1 (Vang et al., 1991;Gu et al., 1992;Wargovich, 2006;Wang et al., 2010Wang et al., , 2015Konstandi et al., 2013;Su et al., 2013;Ahmad et al., 2014;Lu et al., 2014;Zhou et al., 2014Zhou et al., , 2015Shayakhmetova et al., 2015;Suzuki et al., 2015;Jiang et al., 2016a,b). ...
Cytochrome P450 (CYP) 2E1 is an important enzyme involved in the metabolism of many endogenous and exogenous compounds. It is essential to evaluate the expression of CYP2E1 in the studies of drug–drug interactions and the screening of drugs, natural products, and foodstuffs. The present work is a feasibility study on the development of immunoassays using a specific and sensitive chicken-sourced anti-CYP2E1 IgY antibody. Cloning, expression, and purification of a recombinant CYP2E1 (mice origin) protein were carried out. Anti-CYP2E1 IgY antibodies were generated by immunizing white Leghorn chickens with purified recombinant CYP2E1 protein and were purified by immune affinity chromatography. The IgY titer attained a peak level (≥1:128,000) after the fifth booster injection. For evaluation of the expression of CYP2E1 in different herbal treatment samples, the mice were treated by oral gavage for 3 days with alcohol (50% 15 mL/kg), acetaminophen (APAP, 300 mg/kg), Cornus officinalis extract (100 mg/kg), Alhagi-honey extract (100 mg/kg), Apocynum venetum extract (100 mg/kg), hyperoside (50 mg/kg), isoquercetin (50 mg/kg), 4-hydroxyphenylacetic acid (50 mg/kg), 3-hydroxyphenylacetic acid (50 mg/kg), and 3,4-hydroxyphenylacetic acid (50 mg/kg). The expression of CYP2E1 was determined by Western blot analysis, immunohistochemistry, ELISA, and immunomagnetic beads (IMBs) using anti-CYP2E1 IgY in liver tissue. The results showed that C. officinalis extract, Alhagi-honey extract, A. venetum extract, hyperoside, isoquercetin, and their xenobiotics 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-hydroxyphenylacetic acid significantly decreased CYP2E1 levels. Alcohol and APAP treatments significantly increased CYP2E1 levels as analyzed with Western blot analysis, immunohistochemistry, and ELISA. The IMB method is suitable for large-scale screening, and it is a rapid screening (20 min) that uses a portable magnet and has no professional requirements for the operator, which makes it useful for on-the-spot analysis. Considering these results, the anti-CYP2E1 IgY could be applied as a novel research tool in screening for the CYP2E1 inhibitor/enhancer.
... Similarly, CYP2E1 was found to increase liver sensitivity toward LPS and inflammatory mediator toxicity and to potentiate LPS-induced oxidative stress in the liver (57,58); therefore, both INH and LPS caused overactivation of CYP2E1, which, in return, intensified their toxicity. CYP2E1 involvement in INH toxicity is not limited just to the liver: Shayakhmetova et al. declared that the induction of CYP2E1 in rat testicular tissues treated with INH resulted in the triggering and accumulation of ROS that led to testicular toxicity, DNA fragmentation, spermatogenesis disturbances, and male infertility (59). CYP2E1 also had a functional role in bile acid increment through activation of the bile acid synthesis cascade (56). ...
Isoniazid (INH) remains the core in tuberculosis management, but serious hepatotoxicity and potentially fatal liver injury still coming alongside with INH consumption. Among numerous theories that have been established to explain INH-induced liver injury, inflammatory stress theory recently widely used to explain the idiosyncrasy. Inflammatory stress usually sensitized tissues to drug's toxic consequences. So that, this study was conducted to verify whether bacterial lipopolysaccharide (LPS) inflammation could has a role in enhancing INH hepatotoxicity. While single INH or LPS administration showed no major toxicity signs, INH/LPS co-treatment intensified liver toxicity. Both blood biomarkers and histological evaluations clearly showed positive signs of severe liver damage accompanied with massive necrosis, inflammatory infiltration and hepatic steatosis. Furthermore, elevated serum levels of bile acid associated with repressions in bile acid synthesis and transport regulatory parameters were observed. Moreover, the principal impact of cytochrome P450 2E1 (CYP2E1) in INH toxicity could be anticipated, as its protein expression showed enormous increment in INH/LPS co-treated animals. Furthermore, CYP2E1 crucial role in the production of reactive oxygen species (ROS) was obviously noticed through repression of hepatic antioxidant parameters. In summary, these results verified that, this LPS-induced inflammation model might proof valuableness which might help in revealing hepatotoxic mechanisms of INH and the crucial role played by CYP2E1 in initiation and propagation of INH-induced liver damage which could be of great value for clinicians in understanding the pathogenesis of drug-induced liver injury (DILI).
... ADRs may happen following a single dosage or extended administration of a drug or can be outcome of use of combination of two or more drugs (Farcas & Bojita, 2009). A lot of work has been done showing association of toxic effects with first-line ATDs in different organs like liver (Saukkonen et al., 2006;Tostmann et al., 2008) kidney (Assendelft, 1998;Covic et al., 1998;Sweetman, 2009) testes (Alp et al., 2012;Bondarenko et al., 2012;Shayakhmetova et al., 2013Shayakhmetova et al., , 2015 ovaries (Steens, 1977), blood (Fenniche et al., 2003) and bone (Isefuku et al., 2001). Till now nobody has worked upon the splenic toxicity associated with the first-line-antitubercular drugs (ATDs). ...
Context:
Isoniazid, rifampicin and pyrazinamide are most reliable and cost-effective remedy for tuberculosis treatment and prophylaxis among first-line anti-tuberculosis (TB) drugs and have a pronounced tendency to cause adverse drug reactions. Hepatotoxicity is well-studied side effect of these drugs but their effects on other organs like spleen and blood are still needed to be explored.
Objective:
To explore the probable outcome of co-administration these three major antitubercular drugs (ATDs), rifampicin, isoniazid and pyrazinamide on spleen, blood and bone marrow.
Materials and methods:
Different parameters were evaluated like lipid peroxidation, glutathione (GSH) and protein content in spleen by spectrophotometric evaluation, hematological evaluation by determining total hemoglobin, total leukocyte count, differential leukocyte count and scanning electron microscopy studies in blood, genotoxicity studied by bone marrow chromosomal analysis and DNA fragmentation. The female rats n = 12 (150-200 g) were grouped as control group orally given saline and toxicant group given INH (30.85 mg/kg b.wt.) + RIF (61.7 mg/kg b.wt.) + PZA (132.65 mg/kg b.wt.) dosage extrapolated from dose that is used in human for 28 d once daily.
Results:
After 28 d-oral co-administration of anti-TB drugs (INH (30.85 mg/kg b.wt.) + RIF (61.7 mg/kg b.wt.) + PZA (132.65 mg/kg b.wt.)), it was revealed that there were an increase thiobarbituric acid reactive substances, decrease in GSH and protein contents in spleen. Marked changes in hematological parameters, DNA fragmentation and chromosomes were also observed.
Conclusion:
This can be concluded from this work that co-administration of first-line ATDs is toxic to spleen and blood also these drugs can cause damage at genetic level.
... The first group served as the control and was given normal saline while the second group received INH, RIF, PZA and ETB in combination. The drugs were dissolved in normal saline and therapeutic doses [INH (5 mg/kg), RIF (10 mg/kg), PZA (15 mg/kg) and ETB (15 mg/kg)] were given by oral gavage three times in a week for 8 consecutive weeks (WHO, 2014;Shayakhmetova et al., 2015). At the end of the experiment, rats were weighed and sacrificed under light ether anaesthesia. ...
... CYP2E1 catalyzes the biotransformation of numerous drugs, including INH. Importantly, CYP2E1-mediated biotransformation of INH can generate toxic reactive metabolites, thus playing a major role in drug metabolism and the pathophysiology of drug-induced liver injury (Aubert et al., 2011;Santos et al., 2013;Shayakhmetova et al., 2015). Human genetic researches have revealed that cytochrome CYP2E1 is involved in INH-induced hepatotoxicity (Humayun et al., 2014). ...
Isoniazid (INH) is an antituberculosis drug associated with idiosyncratic liver injury in susceptible patients. INH-induced hepatotoxicity remains a significant clinical problem, but the underlying mechanisms are still unclear, despite the growing evidence that INH and/or its major metabolite, hydrazine, play an important role in hepatotoxicity. Copyright (c) 2015 John Wiley & Sons, Ltd. Isoniazid (INH) is an antituberculosis drug associated with idiosyncratic liver injury in susceptible patients. INH-induced hepatotoxicity remains a significant clinical problem, but the underlying mechanisms are still unclear, despite the growing evidence that INH and/or its major metabolite, hydrazine, play an important role in hepatotoxicity.
Fluoride and aluminum are ubiquitous toxic metals with adverse reproductive effects. The citrus flavonoid hesperidin has protective activities but poor solubility and bioavailability. Nanoparticulate delivery systems can improve flavonoid effectiveness. We conducted this study to prepare a pH-responsive chitosan-based nanogel for hesperidin delivery and evaluate its effectiveness against sodium fluoride (NaF) and aluminum chloride (AlCl3) induced testicular toxicity in mice. The nanogel was synthesized using 2 kGy gamma irradiation, enabling a size under 200 nm and enhanced hesperidin release at pH 6 matching testicular acidity. Male mice received 200 mg/kg AlCl3 and 10 mg/kg NaF daily for 30 days. Hesperidin nanogel at 20 mg/kg was administered orally either prophylactically (pretreatment) or after intoxication (posttreatment). The results showed that AlCl3 + NaF induced severe oxidative stress, hormonal disturbance, apoptosis, and endoplasmic reticulum stress, evidenced by significant changes in the studied parameters and testicular histological damage. Hesperidin nanogel administration significantly inhibited oxidative stress markers, restored luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels, and alleviated tissue damage compared to the intoxicated group. It also downregulated the expression level of pro-apoptotic genes Bax, caspase-3, caspase-9, and P38MAPK, while upregulating the expression level of the anti-apoptotic BCL2 gene. Endoplasmic reticulum stress sensors PERK, ATF6, and IRE-α were also downregulated by the nanogel. The chitosan-based nanogel enhanced the delivery and efficacy of poorly bioavailable hesperidin, exhibiting remarkable protective effects against AlCl3 and NaF reproductive toxicity. This innovative nanosystem represents a promising approach to harnessing bioactive phytochemicals with delivery challenges, enabling protective effects against chemical-induced testicular damage.
Graphical Abstract
The associations of organochlorine pesticides (OCPs) with semen quality from human studies are conflicting, and also it is largely unknown whether the associations are modified by genetic polymorphisms. We aimed to evaluate the associations between serum concentrations of 18 OCPs and semen quality among 387 Chinese men, and further to examine the modifying effects by genetic polymorphisms in cytochrome P450 (CYP2E1) and glutathione S-transferase (GSTT1). Multivariable linear regressions were used to evaluate the relationships between serum OCP concentrations and semen quality, and the role of CYP2E1 and GSTT1 polymorphisms in modifying the associations were assessed. Multiple testing was adjusted using the false discovery rate (FDR). We observed that men with detectable concentrations of serum ɤ-HCH had a decrease in sperm motility of 7.07% (95% CI: -10.9%, -3.24%) compared to those with undetectable concentrations (FDR-P value = 0.02). Men with TT of CYP2E1 rs 915906 genotypes had higher median concentrations of serum dieldrin compared with those with CT/CC of CYP2E1 rs 915906 genotypes. There were interactions between CYP2E1 and GSTT1 polymorphisms and certain OCPs namely ɤ-HCH, δ-HCH, dieldrin, endosulfan I, and endrin aldehyde on semen quality. For example, elevated dieldrin levels in relation to decreased sperm concentration, sperm count, and sperm motility were only observed among men with CC of CYP2E1 rs2031920 genotypes (all Pinteraction < 0.05). However, these interactions were not statistically significant after the FDR adjustment. Our results suggested that CYP2E1 and GSTT1 polymorphisms may modify the effects of OCP exposures on semen quality. Due to the relatively small size samples, further investigation is warranted to confirm the findings.
The use of animal models for scientific research is an ancient practice in the biological field, as we are dependent on animals for in vivo experiments. Out of many emerging models that have been developed for different purposes, one to be chosen for the investigation of a specific drug is still a question to many. This chapter provides an insight into the different animal models that have been standardized by scientists for the adverse drug reactions (ADRs) studies, specifically for widely used drugs like NSAIDS, antitubercular drugs, tetracyclines, immunosuppressants, antithyroids, and antiepileptic drugs. This chapter also describes the most common ADRs caused by different drugs. The information gained from this chapter can provide researchers assistance for making a preferable selection of suitable animal models for research purposes.
Isoniazid (INH), a synthetic first-line tuberculosis antibiotic, has been widely used in clinical treatment. It has been reported to cause toxic effects at multiple tissue sites and also increases the incidence of adverse pregnancy outcomes; but the mechanism of action of INH on the reproductive system of female mammals remains unclear. Here, we demonstrate that oral INH (40 mg/kg/day every other day for 28 days) severely affects oocyte maturation and fertilization, late blastocyst development and fertility. We found that INH could disrupt standard spindle assembly, chromosome arrangement, and actin filament dynamics, which compromised meiotic progression of mouse oocytes. INH treatment increased the level of reactive oxygen species (ROS) and activated the oxidative stress response pathway, Keap1-Nrf2. It also caused apoptosis of oocytes and mitochondrial dysfunction. Our findings demonstrate that oral INH reduces fertility and damages the mammalian reproductive system by altering cytoskeletal dynamics and Juno expression, inducing oxidative stress and apoptosis, and activating the Keap1-Nrf2 signaling pathway in mouse oocytes.
First-line antituberculosis drugs, namely, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), contribute to diverse pathological complications. Testicular toxicity is one such complication. Berberis aristata DC is an herb with potentially curative characteristics. The aim of this study was to test whether extract of Berberis aristata DC (Berberidaceae) has curing potential against testicular toxicity. Characterization of extract was done using ultra-performance liquid chromatography along with acute toxicity testing. Antioxidant activity of extract was checked by DPPH inhibition assay and H2O2 scavenging assay. Rats were dosed once daily for 28 days in groups: control group (saline), toxicant group (30.85 mg/kg body weight INH + 61.7 mg/kg body weight RIF + 132.65 mg/kg body weight PZA), treatment groups (TB drugs + 150/300 mg/kg body weight extract) and standard group (TB drugs +100 mg/kg body weight silymarin). Spectrophotometric evaluations of lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione-S-transferase (GST), and catalase (CAT) content in testes were done using standard protocols. DNA fragmentation and histopathological studies were performed to check the damage at the cellular level. Acute toxicity studies revealed LD50 > 5 g/Kg body weight of B. aristata extract. IC50 for DPPH free-radical scavenging activity and H2O2 scavenging assay were 44.78 µg/mL and 85.28 µg/mL, respectively. Results revealed significant increase in thiobarbituric acid reactive substances, decrease in glutathione and different antioxidants levels, DNA fragmentation pattern, and changes in histology in toxicant group. All the changes were absent in high-dose (300 mg/kg body weight) extract treatment group. This work proved that B. aristata extract has protective efficacy against testicular damage caused by anti-TB drugs.
Hydrazide group (-C(O)-NH-NH2) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs), Metabolite Intermediate Complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs / MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM – isonicotinoyl radical. The first cycle requires ~11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ~7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps – (i) N-H bond activation, (ii) loss of N2 molecule and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring / the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug-drug interactions. This QC, molecular docking and QM/MM study explored all the above reaction pathways, established the 3D structures of the RMs and MICs.
Background
Mitochondrion, a double-membrane bound organelle, plays a critical role in eukaryotic cells energy production. Further to its role as ATP-producing factory, mitochondrion has distinct role in a number of cellular functions including calcium and redox signaling, apoptosis, and autophagy. Extensive studies showed the crucial role of mitochondrial dysfunction in pathophysiology of different diseases such as cardiovascular diseases, neurodegenerative diseases, among others. Recently extensive evidences report the promising effects of omega-3 polyunsaturated fatty acids on mitochondrial structure and functions as well as mitochondrial diseases.
Scope and approach
In this paper, we critically review the available evidences on beneficial role of omega-3 polyunsaturated fatty acids on mitochondrial dynamic and biogenesis. We also discuss about chemistry, source, and bioavailability of omega-3 polyunsaturated fatty acids.
Key findings and conclusions
These findings lead to an upsurge of interest in finding a new effective therapeutic strategy based on targeting mitochondrial dysfunction for above-mentioned diseases. Recently, a great body of evidences obtained from experimental studies shows the promising role of omega-3 polyunsaturated fatty acids on mitochondrial function and dynamics. Different clinical trials have also addressed the promising role of omega-3 polyunsaturated fatty acids on different disease and these facts may be related to its favorable effects on mitochondria.
With the rise of multiple-drug resistant tuberculosis (MDR-TB) and extensive drug resistant tuberculosis (XDR-TB), varieties of anti-TB drug combinations and doses have been used clinically worldwide. Long-term therapy with one or more anti-TB drugs and their concomitant use with other medications such as retroviral drugs and schizophrenia medications have resulted in many adverse effects. This chapter mainly focuses on summarizing new and emerging adverse effects of anti-mycobacterial drugs reported in the year of 2015. While common side effects of drugs are briefly covered in this section, the focus was on summarizing rare adverse effects of drugs and mechanisms and genetic predispositions associated with toxicity of selected drugs. Drugs covered in this chapter include rifamycins, bedaquiline, linezolid, isoniazid, ethambutol, fluoroquinolones, pyrazinamide, clofazimine, cycloserine, delamanid, dapsone and streptomycin.
Furan is produced in a wide variety of heat-treated foods via thermal degradation. Furan contamination is found to be relatively high in processed baby foods, cereal products, fruits juices, and canned vegetables. Several studies have demonstrated that furan is a potent hepatotoxin and hepatocarcinogen in rodents. However, few studies have investigated the toxic effects of furan in the testis. In addition, the exact mechanism(s) by which furan exerts toxicity in the testis has not been fully elucidated. In this study, we investigated the potential of furan exposure from weaning through adulthood to induce oxidative stress in adult rat testis, as well as the potential of garlic oil (GO) to ameliorate the induced toxicity. Our results reveal that furan administration significantly reduced serum testosterone levels and increased the levels of malondialdehyde (MDA); furthermore, furan administration decreased significantly the enzymatic activity of testicular antioxidants, including glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and induced histopathological alterations in the testis. GO co-administration ameliorated the reduction in testosterone levels and dramatically attenuated the furan-induced oxidative and histopathological changes. In addition, Go significantly down-regulated the increased caspase-3 and cytochrome P450 2E1 (CYP2E1) expression in the furan-treated testis. To the best of our knowledge, this study is the first to demonstrate the furan-induced oxidative changes in the adult rat testis and the protective role of GO to ameliorate these changes through its antioxidant effects and its ability to inhibit CYP2E1 production.
Diabetes mellitus is one of the three most common modem diseases. A number of animal models is used in investigations of the mechanisms of development of the disease. Most of these models replicate the symptoms of type 1 diabetes mellitus. The development of type 2 diabetes is caused by the insulin resistance, hyperglycemia, structural and functional disorders of the pancreatic cells. Investigation of pathogenesis of type 2 diabetes is complicated by the lack of adequate models of this disease. In this work, based on existing hyperglycemia model, we propose the model of metabolic syndrome as a precursor of type 2 diabetes. The development of metabolic syndrome symptoms was caused by 28 days long intramuscular injection of protamine sulfate to guinea pigs at a dose of 15 mg/kg along with keeping of animals on a high glucose diet. Increased blood glucose and cholesterol levels, reduction of glycogen in liver, the structural and functional damage and reduce in the number of functionally active beta-cells in the pancreas of the experimental animals were observed. The results confirm the development of the metabolic syndrome symptoms in experimental animals, which makes it possible to use such methodical approach in creation of promising type 2 diabetes model.
The purification of homogeneous glutathione S-transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2-dichloro-4-nitrobenzene. Although each of the four enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p-nitrobenzyl chloride. Assay conditions for a variety of substrates are included.
All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite the similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
The rate of autoxidation of epinephrine and the sensitivity of this autoxidation to inhibition by superoxide dismutase were both augmented, as the pH was raised from 7.8 → 10.2. O2⁻, generated by the xanthine oxidase reaction, caused the oxidation of epinephrine to adrenochrome and the yield of adrenochrome produced per O2⁻ introduced, increased with increasing pH in the range 7.8 → 10.2 and also increased with increasing concentration of epinephrine. These results, in conjunction with complexities in the kinetics of adrenochrome accumulation, lead to the proposal that the autoxidation of epinephrine proceeds by at least two distinct pathways, only one of which is a free radical chain reaction involving O2⁻ and hence inhibitable by superoxide dismutase. This chain reaction accounted for a progressively greater fraction of the total oxidation as the pH was raised. The ability of superoxide dismutase to inhibit the autoxidation of epinephrine at pH 10.2 has been used as the basis of a convenient and sensitive assay for this enzyme.
(Y1-xLax)2O3 (x = 00.125) transparent ceramics were fabricated by the conventional ceramics processing. The microstructure, hardness, light transmittance and thermal conductivity of the ceramics was investigated. The results show that La2O3 can effectively promote sintering, and inhibit excessive grain growth. After La2O3 doping, the hardness of the transparent ceramics increases from 786 MPa to 878 MPa, but the thermal conductivity decreases significantly from 16.92 W/(m•K) to 5.68 W/(m•K). The (Y0.90La0.10)2O3 transparent ceramics has excellent optical property, the transmission range is 300–8 500 nm, and the maximum transmittance is greater than 80%.
Tuberculosis (TB) is one of the oldest, the most expanded and the most lethal diseases in human history. Although Koch's discovery of the TB causative agent (1882) represented a great progress in the fight against this infectious disease, it took a significant amount of time to reduce morbidity and mortality in the world. In Bosnia and Herzegovina (B&H) it was necessary to implement actions in the form of education where the popularization of measures of recognition, treatment and preventing the disease was done. After the Annexation of B&H to Austro-Hungarian (1908) began an organized fight against TB. Dr. S. Kukric was a particularly prominent individual, putting his effort by working in clinics, through his lectures and numerous popular research papers on tuberculosis. He was followed by many colleagues working inexhaustibly while facing the high incidence of TB and the difficult social situation in B&H. Although this disease is old, at the end of the 20th century a new TB appeared, with new challenges and new, even grater problems. Significant achievement and great progress in the treatment and control of TB infection was achieved by implementing the direct observed short course treatment (DOTS from 2006.) Still, there is a too high incidence of TB that becomes again a serious threat, together with new problems and difficult economic and social situation. Nowadays in Federation of B&H the guidelines of World Health Organization on reinforcing the DOTS strategy are being daily implemented, including multidrug-resistant (MDR) TB and infection control.
SETTING: Isoniazid (INH) is related to the development of hepatotoxicity in some patients.
OBJECTIVE: To investigate the role of N-acetyl transferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) in the hepatotoxicity of patients treated with INH in an Amazonian Brazilian population.
DESIGN: Patients undergoing anti-tuberculosis treatment were investigated. Hepatotoxicity was defined as an increase of more than three times the upper limit of normal in serum alanine aminotransferase levels after treatment. NAT2 genotypes were identified by sequencing, whereas CYP2E1 alleles were detected using polymerase chain reaction based methods.
RESULTS: Of the 270 individuals included in the study, 18 (6.7%) developed drug-related hepatotoxicity. A high association was found between slow acetylators and hepatotoxicity, particularly with regard to allele *5. The adjusted risk of developing hepatotoxicity was significant in individuals carrying two slow acetylation alleles (P = 0.036, OR 3.05, 95%CI 1.07–8.64). In all of the CYP2E1 markers examined, wild homozygous genotypes were more prevalent in subjects with hepatotoxicity than in controls; however, the difference was not statistically significant. Joint evaluation of the genes revealed a high risk of developing hepatotoxicity in slow acetylators with CYP2E1 wild alleles (adjusted OR 4.26; 95%CI 1.47–12.37, P = 0.008).
CONCLUSIONS: Large-scale screening for NAT2 and CYP2E1 genotypes can prove useful in predicting the risk of adverse effects.
The necessity to minimize adverse effects of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antituberculosis drugs on the reproductive system and testicular cell macromolecules. The epidemiological situation of tuberculosis in Central and Eastern Europe is getting worse. Data on adverse effects of antituberculosis drugs are scare concerning particularly their effects on the reproductive system. The aim of the present study was to investigate the potential effect of ethambutol, rifampicin, isoniazid and pyrazinamide co-administration on lipid peroxidation, glutathione content and protein SH-groups, DNA fragmentation levels, the reproductive capacity of Wistar male rats and the antenatal development of their posterity. The rats (150-170 g) were divided into two groups: group I - received antituberculosis drugs suspended in 1% starch gel per os: ethambutol - 155 mg/kg b.w./day, rifampicin - 74.4 mg/kg b.w./day, isoniazid - 62 mg/kg b.w./day, pyrazinamide - 217 mg/kg b.w./day, group II (control) - received only starch gel in corresponding volumes. The contents of TBA-active compounds, glutathione and protein SH-groups in testis and sperm were determined spectrophotometrically, the DNA-fragmentation was determined using an UV transilluminator (BIORAD, USA), reproductive system indices were measured by standard methods. The co-administration of therapeutic doses of ethambutol, isoniazid, rifampicin and pyrazinamide to male rats during the period of spermatogenesis caused an increase in the rate of thiobarbituric acid reactive substances formation in testis and sperm, decrease of testis glutathione and protein SH-group contents, significant changes in DNA fragmentation, fatal decrease of male fertilizing capacity and fertility, and increase of pre- and post-implantation embryo lethality. The changes in reproductive indices could be the result of direct or indirect effects of one or more drugs investigated.
The key to successful elimination of tuberculosis (TB) is treatment of cases with optimum chemotherapy. Poor chemotherapy over time has led to drug-resistant disease. Drug resistance of Mycobacterium tuberculosis develops by the selective growth of resistant mutants. The incidence of drug-resistant cases depends on the number of bacilli and the drug-resistant mutants in the lesion. The latter is low for individual drugs and even lower for two and three drugs. Therefore, use of combination chemotherapy with three or more drugs results in cure. However, irregular treatment, inadequate drugs, inadequate drug doses or addition of a single drug to a failing regimen allows selective growth of resistant mutants and acquired drug-resistant TB. Contacts of these resistant cases develop primary drug resistant TB. Thus, drug resistance in tuberculosis is a "man-made problem". Anti-TB chemotherapy must be given optimally by (i) ensuring adequate absorption of drugs, (ii) timely diagnosis and management of drug toxicities and (iii) treatment adherence. New classes of anti-TB drugs are needed; but are unlikely to become available soon. It is vital that the 21(st) century physicians understand the basic principles of TB chemotherapy to ensure efficient use of available drugs to postpone or even reverse epidemics drug-resistant TB.
Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca²+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca²+ chelator, abolished Cd-induced [Ca²+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca²+ chelator EGTA also prevented Cd-induced [Ca²+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca²+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca²+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca²+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca²+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.
Oxidative stress (OS) has been considered a major contributory factor to the infertility. Oxidative stress is the result of imbalance between the reactive oxygen species (ROS) and antioxidants in the body which can lead to sperm damage, deformity, and eventually male infertility. Although high concentrations of the ROS cause sperm pathology (ATP depletion) leading to insufficient axonemal phosphorylation, lipid peroxidation, and loss of motility and viability but, many evidences demonstrate that low and controlled concentrations of these ROS play an important role in sperm physiological processes such as capacitation, acrosome reaction, and signaling processes to ensure fertilization. The supplementation of a cryopreservation extender with antioxidant has been shown to provide a cryoprotective effect on mammalian sperm quality. This paper reviews the impacts of oxidative stress and reactive oxygen species on spermatozoa functions, causes of ROS generation, and antioxidative strategies to reduce OS. In addition, we also highlight the emerging concept of utilizing OS as a tool of contraception.
Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.
Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52 mM). Hydrogen peroxide (0.3 mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.
The high incidence of low sperm counts in young (European) men and evidence for declining sperm counts in recent decades mean that the environmental/lifestyle impact on spermatogenesis is an important health issue. This review assesses potential causes involving adverse effects on testis development in perinatal life (primarily effects on Sertoli cell number), which are probably irreversible, or effects on the process of spermatogenesis in adulthood, which are probably mainly reversible. Several lifestyle-related (obesity, smoking) and environmental (exposure to traffic exhaust fumes, dioxins, combustion products) factors appear to negatively affect both the perinatal and adult testes, emphasizing the importance of environmental/lifestyle impacts throughout the life course. Apart from this, public concern about adverse effects of environmental chemicals (ECs) (pesticides, food additives, persistent pollutants such as DDT, polychlorinated biphenyls) on spermatogenesis in adult men are, in general, not supported by the available data for humans. Where adverse effects of ECs have been shown, they are usually in an occupational setting rather than applying to the general population. In contrast, a modern Western lifestyle (sedentary work/lifestyle, obesity) is potentially damaging to sperm production. Spermatogenesis in normal men is poorly organized and inefficient so that men are poorly placed to cope with environmental/lifestyle insults.
Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here, we show that hydrogen peroxide (H(2)O(2)), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H(2)O(2) inhibition of mTOR signaling. Ectopic expression of wild-type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H(2)O(2) induction of apoptosis. Furthermore, we identified that H(2)O(2) induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase alpha (AMPKalpha), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKalpha or downregulation of AMPKalpha1 conferred partial resistance to H(2)O(2) inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H(2)O(2) inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H(2)O(2)-induced neurodegenerative diseases.
Spermatogenesis is an extremely active replicative process capable of generating approxi mately 1,000 sperm a second. The high rates of cell division inherent in this process imply correspondingly high rates of mitochondrial oxygen consumption by the germinal epithelium. However, the poor vascularization of the testes means that oxygen tensions in this tissue are low1 and that competition for this vital element within the testes is extremely intense. Since both spermatogenesis2 and Leydig cell steroidogenesis3,4 are vulnerable to oxidative stress, the low oxygen tension that characterizes this tissue may be an important component of the mechanisms by which the testes protects itself from free radical-mediated damage. In addition, the testes contain an elaborate array of antioxidant enzymes and free radical scavengers to ensure that the twin spermatogenic and steroidogenic functions of this organ are not impacted by oxidative stress. These antioxidant defence systems are of major importance because peroxidative damage is currently regarded as the single most important cause of impaired testicular function underpinning the pathological consequences of a wide range of conditions from testicular torsion to diabetes and xenobiotic exposure. This chapter sets out the specific nature of these antioxidant defence systems and also reviews the factors that have been found to impair their activity, precipitating a state of oxidative stress in the testes and impairing the latter’s ability to produce viable spermatozoa capable of initiating and supporting embryonic development.
Spermatogenesis is a complex process involving mitotic cell division, meiosis and the process of spermiogenesis. The regulation
of spermatogenesis involves both endocrine and paracrine mechanisms. The endocrine stimulation of spermatogenesis involves
both follicle stimulating hormone (FSH) and luteinizing hormone, the latter acting through the intermediary testosterone,
produced by the Leydig cells in the testis. Since the germ cells do not possess receptors for FSH and testosterone, the hormonal
signals are transduced through the Sertoli cells and peritubular cells by the production of signals that have yet to be defined.
Although the hormonal signals are essential for successful spermatogenesis, there is increasing evidence that a multiplicity
of growth factors and cytokines are involved in local control mechanisms influencing stem cell renewal by mitosis and the
complicated process of the two meiotic cell divisions. The final complex metamorphosis which converts a round cell into the
complex structures of the spermatozoa is well defined at a structural level, but the control systems regulating this process
still remain to be elucidated.
Isoniazid (INH) remains the most safe and cost-effective drug for the treatment and prophylaxis of tuberculosis. The use of INH has increased over the past years, largely as a result of the coepidemic of human immunodeficiency virus infection. It is frequently given chronically to critically ill patients who are coprescribed multiple medications. The ability of INH to elevate the concentrations in plasma and/or toxicity of coadministered drugs, including those of narrow therapeutic range (e.g., phenytoin), has been documented in humans, but the mechanisms involved are not well understood. Using human liver microsomes (HLMs), we tested the inhibitory effect of INH on the activity of common drug-metabolizing human cytochrome P450 (CYP450) isoforms using isoform-specific substrate probe reactions. Incubation experiments were performed at a single concentration of each substrate probe at its K(m) value with a range of INH concentrations. CYP2C19 and CYP3A were inhibited potently by INH in a concentration-dependent manner. At 50 microM INH (approximately 6.86 microg/ml), the activities of these isoforms decreased by approximately 40%. INH did not show significant inhibition (<10% at 50 microM) of other isoforms (CYP2C9, CYP1A2, and CYP2D6). To accurately estimate the inhibition constants (K(i) values) for each isoform, four concentrations of INH were incubated across a range of five concentrations of specific substrate probes. The mean K(i) values (+/- standard deviation) for the inhibition of CYP2C19 by INH in HLMs and recombinant human CYP2C19 were 25.4 +/- 6.2 and 13 +/- 2.4 microM, respectively. INH showed potent noncompetitive inhibition of CYP3A (K(i) = 51.8 +/- 2.5 to 75.9 +/- 7.8 microM, depending on the substrate used). INH was a weak noncompetitive inhibitor of CYP2E1 (K(i) = 110 +/- 33 microM) and a competitive inhibitor of CYP2D6 (K(i) = 126 +/- 23 microM), but the mean K(i) values for the inhibition of CYP2C9 and CYP1A2 were above 500 microM. Inhibition of one or both CYP2C19 and CYP3A isoforms is the likely mechanism by which INH slows the elimination of coadministered drugs, including phenytoin, carbamazepine, diazepam, triazolam, and primidone. Slow acetylators of INH may be at greater risk for adverse drug interactions, as the degree of inhibition was concentration dependent. These data provide a rational basis for understanding drug interaction with INH and predict that other drugs metabolized by these two enzymes may also interact.
Dysregulation of male germ cell apoptosis has been associated with the pathogenesis of male infertility. Therefore, factors involved in the regulation of germ cell death are being actively investigated. Here, we studied the effects of lactate on human male germ cell death, using as a model a testis tissue culture in which physiological contacts are maintained between the germ cells and the supportive somatic Sertoli cells. Apoptosis of spermatocytes, spermatids and a few spermatogonia was induced by culturing segments of seminiferous tubules under serum-free conditions. This germ cell death was inhibited effectively and dose-dependently by lactate, indicating that it plays a crucial role in controlling cell death cascades of male germ cells. Interestingly, the anti-apoptotic role of lactate was not associated with changes in testicular adenine nucleotide (ATP, ADP and AMP) levels. In the seminiferous tubules, the final site of the death-suppressing action of lactate appeared to be downstream along the cell death pathway activated by the Fas receptor of the germ cells. In conclusion, testicular cell death was effectively regulated by lactate, which may be regarded as a potential compound for optimizing in-vitro methods involving male germ cells for assisted reproduction.
Animals. Use Sexually Mature Animals, Whenever Practical, for Evaluating Effects on Spermatogenesis. Unless the objective of the study dictates otherwise, screening studies in rodents should be conducted in animals that are sexually mature at termination of the study (>9 weeks in rat, >7 weeks in mouse). Ideally dogs should be 9-12 months of age at termination, to minimize confounding aspects of immaturity. If younger animals are used and equivocal results occur, it may be necessary to repeat the study in older animals. The difficulties in obtaining sexually mature primates and the associated handling hazards for laboratory personnel generally preclude their use in screening studies and therefore preclude assessment of treatment-related effects on spermatogenesis. For suspected testicular toxicants, specific studies in sexually mature animals should be considered (4-5 years of age for cynomolgous). Tissue Sampling Sample. Both Testes and Epididymides, Identify Left and Right Separately and Record Organ Weights. Distinction of unilateral from bilateral effects is important for evaluation of treatment related findings, therefore separating data sets for each side can provide important information. Histopathological findings in the testis and epididymis are often interelated; it is useful to be able to directly correlate such findings. Increases and decreases in testis and epididymal weight are important indicators of adverse effects. Tissue Fixation. Fix Testes from All Species from Studies of 13 Weeks Duration and Less in Modified Davidson's Fixative. Well-fixed tissue is critical for the detection of early and subtle changes in the testis. Regulatory guidelines discourage the use of formalin for testes and recommend Bouin's or an alternative. Modified Davidson's provides improved fixation properties over Bouin's and is significantly less hazardous and more convenient to use on a routine basis. The design of fertility studies relies on information gained from examination of the testes in general toxicity studies. Therefore, adequate fixation in all studies up to 13-weeks' duration is recommended. Tissue Trimming, Sectioning, and Staining. Embed Tissues in Paraffin Wax. Embed Transverse Sections of the Testes, to Include Part of the Rete. Embed Longitudinal Sections of the Epididymis, Incorporating the Caput, Corpus and Cauda. For Rodent Studies up to 28 Days, Examine PAS-H-Stained Testes. For Rodent Studies Over 28 Days and for All Dog and Primate Testes, Examine H&E Stained Testes. Wax sections provide adequate quality for evaluating screening studies. Resin embedding may be used for investigative studies. Transverse sections provide mostly round, cross-sectional profiles of tubules for evaluation. The rete is a potential site for compound-related findings. The various regions of the epididymis have different functions, morphology, and susceptibility for toxicity. The lumenal contents of the different regions of the epididymis also reflect different temporal events in the testes. PAS-H stained sections allows accurate identification of the tubular stage of spermatogenesis in rats and mice. This information is useful for identifying and evaluating early disturbances of spermatogenesis in studies of short duration (up to 28 days). PAS-H is less useful in the dog and primate due to its restricted staining of the acrosome. Microscopic Evaluation. Microscopic Evaluation of the Testis Should be Carried Out as a Qualitative Examination with an Awareness of the Spermatogenic Cycle. Recognition of the loss of specific populations of germ cells or the inappropriate presence of germ cells (eg, spermatid retention) relies on knowledge of the cellular associations of the different stages of the spermatogenic cycle. The pathologist must have an adequate understanding of the morphology of the different stages of the cycle in the species under investigation. An understanding of the kinetics of the cycle as well as the physiology and regulation of spermatogenesis is also necessary to allow interpretation of the patterns of changes seen and their toxicological significance. Quantitative measures of tubular stages and their frequency distribution are not recommended for screening studies. Nomenclature and Severity Grading for Disturbances in Spermatogenesis May Vary on a Case-by-Case Basis Depending on the Specificity of the Findings. Findings may be nonspecific and be adequately described by nonspecific terminology such as tubular atrophy. This is frequently the case following long-term exposure to a toxicant. Conversely they may be cell specific and stage specific and require detailed terminology to convey this specificity. This is more often the case in short-duration studies. Grading severity may be based on the proportion of tubules affected or on the proportion of germ cells affected or lost. The choice is dependent on the changes seen and the nomenclature used. Whatever system is used, it is important that the diagnostic and grading criteria are defined.
To study the effect of bisphenol A on the epididymis and epididymal sperm of rats and the possible amelioration action of co-administration with vitamin C.
Male Wistar rats were orally administered bisphenol A (0.2 microg x kg (-1) x day(-1), 2 microg x kg(-1) x day(-1) and 20 microg x kg(-1) x day(-1)) and 0.2 microg, 2 microg and 20 microg bisphenol A + 40 mg vitamin C x kg(-1) x day(-1) for 60 days. On day 61, rats were killed with anesthetic ether and sperm collected from epididymis were used or assessment of sperm count, motility and viability and biochemical studies. A 1 % homogenate of epididymis was prepared and used for biochemical estimations. Caput, corpus and cauda epididymis were fixed in Bouin's fixative for histological studies.
Administration of bisphenol A caused a reduction in the epididymal sperm motility and count and the sperm viability remained unchanged. The activities of superoxide dismutase and glutathione peroxidase decreased, while the levels of lipid peroxidation increased in epididymal sperm and epididymis at all doses. Co-administration with vitamin C reversed the effect of bisphenol A-induced oxidative stress in epididymal sperm and epididymis. A complete degeneration of epididymal epithelium in caput, corpus and cauda regions with reduction in the number of sperms were observed at all doses of bisphenol A-treated rats.
Bisphenol A induced oxidative stress in epididymis and caused degeneration of the epididymal epithelium of rats. Co-administration with vitamin C had a protective effect against the bisphenol A-induced toxicity in epididymal sperm and epididymis.
There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.
Psychiatric nurses have long advocated for children with mental disorders, but few have gone behind bars to advocate for these youth. This paper offers suggestions for advocating on behalf of the most underserved of children?youthful offenders. Efforts in Maryland, as an example, portray conflicting convictions: to punish or to treat. These conflicting convictions permeate our juvenile laws, juvenile facilities, and treatment programs. A discussion of successful programming demonstrates the strengths of advocacy and the difference one person can make. Steps are outlined to assist nurses in the development of activist roles in advocating for these troubled youth.
Dysregulation of male germ cell apoptosis has been associated with the pathogenesis of male infertility. Therefore, factors involved in the regulation of germ cell death are being actively investigated. Here, we studied the effects of lactate on human male germ cell death, using as a model a testis tissue culture in which physiological contacts are maintained between the germ cells and the supportive somatic Sertoli cells. Apoptosis of spermatocytes, spermatids and a few spermatogonia was induced by culturing segments of seminiferous tubules under serum-free conditions. This germ cell death was inhibited effectively and dose-dependently by lactate, indicating that it plays a crucial role in controlling cell death cascades of male germ cells. Interestingly, the anti-apoptotic role of lactate was not associated with changes in testicular adenine nucleotide (ATP, ADP and AMP) levels. In the seminiferous tubules, the final site of the death-suppressing action of lactate appeared to be downstream along the cell death pathway activated by the Fas receptor of the germ cells. In conclusion, testicular cell death was effectively regulated by lactate, which may be regarded as a potential compound for optimizing in-vitro methods involving male germ cells for assisted reproduction.
Tuberculosis (TB), an ongoing public health threat, is worsened by the emergence of drug resistance. With an estimated 630000 cases per year of multidrug resistant (MDR)-TB, and 9% of those being extensively drug resistant (XDR)-TB, there is an urgent need for new and more effective anti-TB drugs. New TB treatment regimens should be able to shorten the duration of therapy that currently takes at least six months. The non-compliance with this long treatment duration is one of the reasons for the development of drug resistance. In spite of the difficulties and alleged lack of interest from the pharmaceutical industry for the discovery and development of new antibiotics, several new or repurposed drugs are being evaluated in clinical trials. This review article summarizes the information available and presents an update on the drugs currently in clinical trials for TB and briefly introduces some new compounds in pre-clinical development.
Unlabelled:
Isoniazid (INH)-induced hepatotoxicity remains one of the most common causes of drug-induced idiosyncratic liver injury and liver failure. This form of liver injury is not believed to be immune-mediated because it is not usually associated with fever or rash, does not recur more rapidly on rechallenge, and previous studies have failed to identify anti-INH antibodies (Abs). In this study, we found Abs present in sera of 15 of 19 cases of INH-induced liver failure. Anti-INH Abs were present in 8 sera; 11 had anti-cytochrome P450 (CYP)2E1 Abs, 14 had Abs against CYP2E1 modified by INH, 14 had anti-CYP3A4 antibodies, and 10 had anti-CYP2C9 Abs. INH was found to form covalent adducts with CYP2E1, CYP3A4, and CYP2C9. None of these Abs were detected in sera from INH-treated controls without significant liver injury. The presence of a range of antidrug and autoAbs has been observed in other drug-induced liver injury that is presumed to be immune mediated.
Conclusion:
These data provide strong evidence that INH induces an immune response that causes INH-induced liver injury.
Background:
The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions.
Scope of review:
The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism.
Major conclusions:
All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle.
General significance:
In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione.
Steroidogenesis decreases with aging in the testis, whereas the levels of reactive oxygen species (ROS) increase. In addition, ROS have been reported to inhibit testicular steroidogenesis. Here, we investigated the effects of ROS on the transcriptional activity of Nur77, one of the major transcription factors that regulate the expression of steroidogenic enzyme genes. ROS signaling inhibited Nur77 transactivation, which was diminished by either treatment with c-Jun N-terminal kinase (JNK) inhibitor or the expression of a dominant negative form of JNK. This suggests the involvement of JNK signaling, which elevates the expression of c-Jun as well as its phosphorylation in Leydig cells. In transient transfection assays, c-Jun suppressed Nur77 transactivation in a dose-dependent manner. Further studies using c-Jun mutants revealed that the protein level of c-Jun, but not phosphorylation itself, was important for the suppression of Nur77 transactivation. Nur77 directly interacted with c-Jun in vivo, which blocked the DNA binding activity of Nur77. Together, these results suggest that ROS signaling-mediated c-Jun upregulation suppresses the expression of steroidogenic enzyme genes by inhibiting Nur77 transactivation, resulting in the reduction of testicular steroidogenesis. These findings may provide a mechanistic explanation for the age-related decline in testicular steroid hormone production.
The speed and position estimation from Back EMF has been a problem under speed reversal response due to change of sign of speed. In this paper, an algorithm using fuzzy logic is proposed to estimate the speed and position of BLDC motor from back EMF for sensorless brushless DC (BLDC) motor drives to improve the performance of conventional sensorless drives. Most existing sensorless methods of the BLDC motor have low performance at speed reversal, transients or low speed range and occasionally require additional circuit. For this type of problem, the estimation of speed and position from back-EMF is suitable for high performance because the back-EMF of the BLDC motor has a trapezoidal shape. The proposed algorithm gives robust control for the reversal of the reference speed and continuously calculates position of the rotor at transients as well as steady state. The robustness of the proposed algorithm is proved through the MATLAB simulation.
As a first-line anti-tuberculosis drug, isoniazid has a serious adverse side effect: hepatotoxicity. Therefore, the assessment and monitoring of hepatotoxicity from isoniazid to prevent liver injury are great concerns. In this experiment, we compared the levels of ALT in plasma and DNA methylation. 30 male SD rats were allocated randomly into two groups, a control group and an isoniazid group, and treated, respectively, with pure water and isoniazid at low dosage (10 mg/(kg day)) for 42 days by oral gavage. Five rats per group were sacrificed after 14, 28, and 42 days of isoniazid treatment. The levels of methylation in the genome and LINE-1 were measured, in which hypomethylation in the whole genome and LINE-1 repetitive sequences was observed in the INH group during the later period of the experiment (the 42nd day) accompanied with pathological changes in the liver. Thus, our results suggest that low dose of isoniazid can induce liver injury and that level of DNA methylation may be a more sensitive marker for monitoring drug-induced hepatotoxicity than aminotransferase.
Small ubiquitin-like modifiers (SUMO) proteins have been implicated in cellular stress response in different tissues, but whether sumoylation has a similar role during spermatogenesis is currently unknown. In this study, changes in the levels of both free SUMO isoforms and high-molecular weight (HMW) SUMO conjugates were monitored before and after the induction of different types of cellular stresses. Using cell lines and primary cells freshly isolated from mouse testes, significant changes were detected in the levels of SUMO1 and SUMO2/3 conjugates following short exposure of the cells to heat stress and oxidative stress. While high concentrations of H(2)O(2) caused an increase in protein sumoylation, low concentrations of H(2)O(2) mostly caused protein desumoylation. Immunofluorescence studies localized SUMO to the sites of DNA double-strand breaks in stressed germ cells and during meiotic recombination. To study the effect of oxidative stress in vivo, animals exposed to tobacco smoke for 12 weeks were used. Changes in sumoylation of HMW proteins were consistent with their oxidative damage in the tobacco-exposed mice. Our results are consistent with the important roles of different SUMO isoforms in stress responses in germ cells. Furthermore, this study identified topoisomerase 2 alpha as one of the targets of sumoylation during normal spermatogenesis and under stress.
The 2 hydrazine derivatives isoniazid (INH) and procarbazine hydrochloride (P) were injected intravenously into rabbits. Radioactive thymidine was injected into both testicles. Rabbits were ejaculated repeatedly, sperms were counted and incorporation of [3H] thymidine into sperm head DNA was determined by liquid scintillation counting. In P-treated rabbits (5 and 50 mg/kg) radioactivity was significantly increased in sperms that were in late phases of spermatocyte and early phases of spermatid maturation at the time of treatment. This indicates that DNA repair synthesis, (unscheduled DNA synthesis, UDS) occurred following drug-induced DNA damage in these germ cells. Normal DNA synthesis in spermatogonia was inhibited by the high dose only. INH (50 and 125 mg/kg) did not cause UDS in spermatocytes and spermatids and did not affect normal DNA synthesis in spermatogonia. The results are in agreement with literature data indicating that P is a potent mutagen and carcinogen. INH, on the other hand, has weak mutagenic and carcinogenic activities that are most apparent in mice.
The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.
These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 +/- 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 +/- 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent Km for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent Km for NADPH was 11 microM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.
The purification of homogeneous glutathione S transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2 dichloro 4 nitrobenzene. Although each of the 4 enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p nitrobenzyl chloride. Assay conditions for a variety of substrates are included. All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
The molecular mechanism of ethanol-inducible cytochrome P450(CYP2E1) induction by isoniazid was studied and compared to that of pyridine, an inducer of CYP2E1. Aniline hydroxylase and immunoreactive CYP2E1 protein were significantly induced by isoniazid without or with only slight activation of other cytochromes P450. In contrast, pyridine increased the activities of a broad range of P450s. The effects of two structural analogs of isoniazid, isonicotinamide and isonicotinic acid were also tested and found to have a markedly decreased ability to induce CYP2E1. The induction of CYP2E1 by isoniazid was not accompanied by an increased level of CYP2E1 mRNA, and was completely blocked by pretreatment with cycloheximide or sodium fluoride, inhibitors of mRNA translation. These data thus suggest that CYP2E1 induction by isoniazid is due to activation of CYP2E1 mRNA translation and that the hydrazide group on the pyridine ring of isoniazid is important both in the selective induction of CYP2E1 and for magnitude of effect.
Alcohol administration results in activation of the hypothalamic-pituitary-adrenal (HPA) axis, with female rats secreting more adrenocorticotropin (ACTH) and corticosterone (B) than males in response to the same dose of alcohol. We first examined the ontogeny of the gender difference in HPA responsiveness to alcohol by administering four doses (0, 1, 2, or 3 g/kg body weight) to animals at 21, 41, and 61 days of age (prepubertal, peripubertal, and postpubertal, respectively). We then investigated the organizational role of steroids by manipulating the neonatal steroidal milieu. Rats of both genders were gonadectomized or injected with testosterone propionate within 24 hr of birth and the HPA response to 3 g/kg body weight alcohol was tested in adulthood (postpubertal period). Our data show that the gender difference in HPA responsiveness to alcohol administration arises peripubertally. In addition, HPA response to alcohol is quantitatively smaller in intact male rats than in feminized groups (gonadectomized males and females, intact females) and masculinized female rats. We conclude that the gender difference in HPA response to alcohol observed in postpubertal rats injected with alcohol depends on the activational role of testicular androgens, rather than on their organizational influence.
The role of the microsomal ethanol-oxidizing system (MEOS) in hepatic ethanol metabolism is reviewed, with focus on its constitutive, ethanol-inducible cytochrome P-4502E1 (2E1). The MEOS was purified and reconstituted using 2E1, phospholipids, and cytochrome P-450 reductase and shown to oxidize ethanol to acetaldehyde, mainly as a monooxygenase and secondarily via hydroxyl radicals, with transcriptional and posttranscriptional regulation. Polymorphism of 2E1 was recognized, and enzymology (including cofactors, role of lipids, inducers, and inhibitors) as well as cellular and tissue distribution were chartered. Physiological functions involve lipid metabolism and ketone utilization in starvation, obesity, and diabetes. The most significant role of 2E1 is its adaptive response to high blood ethanol levels with a corresponding acceleration of ethanol metabolism. The associated free radical production, however, contributes to liver injury in the alcoholic. Most importantly, 2E1 has a unique capacity to activate many xenobiotics (85 of which are listed) to hepatotoxic or carcinogenic products. Induction of 2E1 also results in enhanced production of acetaldehyde, a highly reactive and toxic metabolite. The proliferation of the endoplasmic reticulum associated with 2E1 induction is also accompanied by enhanced activity of other cytochrome P-450s, resulting in accelerated metabolism of, and tolerance to, other drugs, as well as increased degradation of retinol and its hepatic depletion. Some substrates and metabolites, however, are innocuous and may eventually be used as markers of heavy drinking. Recently discovered effective 2E1 inhibitors also have great therapeutic potential.
Cytochrome P450 2E1 participates in the bioactivation of a wide variety of environmental and occupational pollutants. Such reactions may lead to the production of active carcinogenic metabolites. The presence of P450 2E1 in the testis and prostate has not yet been reported. In the present study, cytochrome P450 2E1 mRNA has been identified in the rat prostate and testis by reverse transcription PCR, southern blotting, and DNA sequencing. P450 2E1 protein from rat testis could be detected with immunoblot analysis, but was not detected in the prostate. The hydroxylation of p-nitrophenol, known to be mediated by P450 2E1, was demonstrated by HPLC measurement of product formation in microsomal fractions from the rat testis, but again not from prostate. Exposure of rats to pyridine resulted in a 2.9-fold increase of p-nitrophenol hydroxylation by testicular microsomes. Diethyldithiocarbamate, a selective mechanism-based inhibitor of P450 2E1, or a P450 2E1 monoclonal antibody, caused marked inhibition of testicular microsomal p-nitrophenol hydroxylase activity. These results indicate that cytochrome P450 2E1 is present in the rat testis, and that it is elevated by the treatment of the animals with pyridine. Thus, the presence and inducibility of cytochrome P450 2E1 in the testis may be of significance in the bioactivation of environmental chemicals to genotoxic metabolites.
The importance of the cytochrome P450 (CYP) enzyme family in xenobiotic metabolism, as well as their differential expression and activity in response to a wide range of environmental chemicals and pharmaceuticals, is well documented. The objective of this study was to evaluate the specificity of the branched DNA (bDNA) signal amplification technique for the detection of multiple rat CYPs from hepatocellular RNA. Oligonucleotide probe sets were designed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3. The robustness of the bDNA assay was assessed with the CYP2B1/2-specific probe set, and total RNA was isolated from control and phenobarbital (PB)-treated rats. Analysis of these RNA samples by bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magnitude (0.1-100 microg of total RNA). The fidelity of the bDNA assay was evaluated within a single assay and between assays where repeated measurements of a single sample were reproduced reliably. The specificity of individual CYP probe sets was evaluated with five typical CYP-inducing chemicals on the expression of specific hepatic CYP mRNA transcripts. Male Sprague-Dawley rats were administered 3-methylcholanthrene, PB, isoniazid, pregnenolone-16alpha-carbonitrile, or clofibric acid to induce transcription of CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of chemical-induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1 and CYP1A2 mRNA levels 670- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; pregnenolone-16alpha-carbonitrile induced CYP3A1/23 expression 34-fold; and clofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data support the use of bDNA signal amplification technology as a robust, reproducible, and efficient means of monitoring the differential expression of multiple isoforms of the CYP enzyme family.
Cytochrome P450 (CYP) 2E1 is a toxicologically important enzyme that inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Although cDNAs for the human, rodent, and rabbit forms of CYP2E1 have been isolated and studied extensively, there is an absence of information about canine CYP2E1, despite the fact that the dog is routinely used in drug safety studies. In this study, we isolated and sequenced a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced canine CYP2E1 amino acid sequence exhibited 75 to 76% identity with rat, mouse, and rabbit CYP2E1 sequences, and 77% identity with human CYP2E1. Two populations of clones, differing at a single nucleotide, were isolated from the unamplified library. The T1453C base change results in a Tyr485His amino acid substitution, which is well beyond the heme binding region but is possibly part of a beta-sheet structure. An allele-specific polymerase chain reaction-based restriction enzyme test was developed for genotyping individual dogs from genomic DNA samples. One hundred mixed breed dogs were genotyped, and the frequencies of the Tyr485 and His485 alleles were found to be 0. 85 and 0.15, respectively. The canine Tyr485 and His485 alleles and human CYP2E1 were expressed in Escherichia coli cells, and catalytic activities of the proteins were assessed using the substrate chlorzoxazone. Although the two canine enzymes had similar catalytic activity; significant kinetic differences were seen between canine and human CYP2E1s.
Induction of cytochrome P450 2E1 (CYP2E1) and the formation of reactive oxygen species (ROS) appear to be one of the mechanisms by which ethanol is hepatotoxic. Glutathione peroxidase and catalase detoxify H(2)O(2). Glutathione S-transferases (GST) provide protection from membrane lipid peroxidation, have GSH peroxidase activity, and reduce lipid hydroperoxides. Previous studies showed an up-regulation of GSH synthesis in CYP2E1 expressing HepG2 cells; this finding prompted an evaluation of the levels of other antioxidant exzymes. In CYP2E1 expressing cells, the alpha and microsomal GST messenger RNA (mRNA) are increased by 4- and 2-fold, respectively, and catalase protein and mRNA is increased by 2-fold. The increase in alpha and microsomal GST mRNA correlates with increased total enzymatic activity and is caused by increased transcription as evidenced by run-on transcription assays. In HepG2 cells transfected to express a different cytochrome P450, CYP3A4, there was an increase in alpha GST. However, in contrast to the CYP2E1 expressing cells, neither microsomal GST nor catalase were induced, suggesting some specificity for CYP2E1. In agreement with an increased antioxidant defense system, the sensitivity to added prooxidants such as menadione, antimycin A, H(2)O(2), and 4-hydroxynonenal was lower in the CYP2E1 expressing cells as compared with control cells. In conclusion, overexpression of CYP2E1 in HepG2 cells, besides elevating total GSH levels, also induces expression of catalase and alpha and microsomal GST. This induction confers resistance to the cells against several prooxidants and is suggested to reflect an adaptive response by the cells against CYP2E1-mediated oxidative stress.
The widespread occupational exposure to trichloroethylene (TCE) led us to test the hypothesis that TCE causes toxicity in the male reproductive system. We also investigated mechanisms mediating the potential cytotoxic response. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for a total of 19 days. Exposure after the first week was interspersed by a "weekend." To estimate internal exposure, we measured the TCE metabolites, trichloroacetic acid (TCA) and trichloroethanol (TCOH), in urine at Days 4, 9, 14, and 19. Urinary excretion of TCOH was significantly higher than TCA; levels of TCOH and TCA significantly increased by the second and third week, respectively. Cytochrome P450 2E1 (CYP2E1), an enzyme involved in TCE metabolism, was localized in the epididymal epithelium and testicular Leydig cells, and was found at higher levels in the former than the latter. Immunoblotting confirmed that CYP2E1 protein was present in greater amounts in epididymis than in testis. p-Nitrophenol hydroxylation, a CYP2E1 catalytic activity, was also higher in the epididymis than in the testis. Chloral, a major TCE metabolite, was generated in microsomal incubations at significantly higher levels in epididymis than in testis. Antibody inhibition of CYP2E1 reduced chloral formation, which was more pronounced in epididymis than in testis. After 4 weeks of TCE exposure, damage to the epididymis was manifested as sloughing of epithelial cells. These results indicated that TCE is metabolized in the male reproductive tract, leading to adverse effects that are more severe in the epididymis than in the testis.
It has been well known that reactive oxygen species (ROS) are produced in the steroidogenic pathway and spermatozoa. H2O2, one of ROS produced by spermatozoa, appears to be a primary toxic agent. In the present study, we examined the effects of H2O2 on the basal and evoked-testosterone release from primary Leydig cells, the protein expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were also investigated. Our preparation was found to contain approximately 87% Leydig cells and very few macrophages. The results demonstrated that H2O2 (>1 x 10(-4) M) significantly inhibited the basal and hCG-stimulated testosterone release. H2O2 abolished forskolin- or 8-Br-cAMP-evoked testosterone release. In the presence of pregnenolone, progesterone, or androstenedione, the inhibitory effect of H2O2 on testosterone release was prevented. H2O2 also inhibited pregnenolone production in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase), therefore diminished the activity of P450scc in Leydig cells. In addition to the inhibition of hormone secretion, H2O2 also regulated steroidogenesis by diminishing protein expression of StAR. These results suggest that H2O2 acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450scc activity and StAR protein expression.