Narayana Kilarkaje

Antioxidants enhance the recovery of three cycles of bleomycin, etoposide, and cisplatin–induced testicular dysfunction, pituitary-testicular axis, and fertility in rats

OBJECTIVE To investigate the effects of an antioxidant cocktail (AC) on bleomycin, etoposide, and cisplatin (BEP)-induced testicular dysfunction. DESIGN In vivo study. SETTING Research laboratory. ANIMAL(S) Adult male and female Sprague-Dawley rats. INTERVENTION(S) The rats were treated with three cycles of 21 days each of therapeutically relevant dose levels of BEP (0.75, 7.5, and 1.5 mg/kg) with or without the AC (a mixture of α-tocopherol, L-ascorbic acid, Zn, and Se). MAIN OUTCOME MEASURE(S) Sperm parameters, fertility, serum hormone levels (ELISA), testicular histopathology, and expression of proliferating cell nuclear antigen (PCNA), and transferrin (Western blotting and immunohistochemistry) were evaluated at the end of treatment and a 63-day recovery period. RESULT(S) At the end of treatment, the AC improved BEP-induced decrease in sperm motility and increase in abnormality but had no effect on reduced sperm count, fertility, and tubular atrophy, although it up-regulated germ cell proliferation. The AC normalized reduced inhibin B levels, but had no effect on decreased transferrin and testosterone and elevated LH levels. At the end of the recovery period, the AC enhanced the expression of PCNA and transferrin, repopulation of germ cells, LH-testosterone axis, and fertility, but had no effect on reduced FSH and elevated inhibin B levels. CONCLUSION(S) The antioxidants protect and then enhance the recovery of testicular and reproductive endocrine functions when administered concomitantly with BEP therapy. The AC may be beneficial to regain testicular functions after chemotherapy.

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13-15 weeks; n=6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P<0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P<0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P<0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction.

Gestational lead exposure induces developmental abnormalities and up-regulates apoptosis of fetal cerebellar cells in rats

Abstract Lead (Pb), a known environmental toxicant, adversely affects almost all organ systems. In this study, we investigated the effects of maternal lead exposure on fetal rat cerebellum. Female Sprague–Dawley rats were given lead nitrate in drinking water (0, 0.5, and 1%) for two weeks before conception, and during pregnancy. Fetuses were collected by caesarian section on gestational day 21 and observed for developmental abnormalities. The fetal cerebellar sections from control and 1% lead group were stained with cresyl violet. Immunohistochemical expressions of p53, Bax, Bcl-2, and caspase 3 were quantified by AnalySIS image analyzer (Life Science, Germany). Lead exposure induced developmental abnormalities of eyes, ear, limbs, neck and ventral abdominal wall; however, these abnormalities were commonly seen in the 1% lead-treated group. In addition, lead also caused fetal mortality and reduced body growth in both dose groups and reduced brain weight in the 1% lead-treated group. The fetal cerebella from the 1% lead-treated group showed unorganized cerebellar cortical layers, and degenerative changes in granule and Purkinje cells such as the formation of clumps of Nissl granules. An increase in Bax and caspase 3, and a decrease in Bcl-2 (p < 0.05), but not in p53, showed apoptosis of the neurons. In conclusion, gestational lead exposure in rats induces fetal toxicity and developmental abnormalities. The lead exposure also impairs development of cerebellar layers, induces structural changes, and apoptosis in the fetal cerebellar cortex. These results suggest that lead exposure during gestation is extremely toxic to developing cerebellum in rats.

Diabetes-Induced Oxidative DNA Damage Alters p53-p21CIP1/Waf1 Signaling in the Rat Testis

Diabetes is increasingly becoming a major cause of large-scale morbidity and mortality. Diabetes-induced oxidative stress alters numerous intracellular signaling pathways. Although testicular dysfunction is a major concern in diabetic men, the mechanistic alterations in the testes that lead to hypogonadism are not yet clear. Oxidative mitochondrial DNA damage, as indicated by 7,8-dihydro-8-oxo-2′-deoxyguanosine, and phosphorylation of p53 at ser315 residue (p-p53ser315) increased in a stage- and cell-specific manner in the testes of rats that were diabetic for 1 month (DM1). Prolongation of diabetes for 3 months (DM3) led to an increase in nuclear oxidative DNA damage in conjunction with a decrease in the expression of p-p53ser315. The nuclei of pachytene and preleptotene spermatocytes, steps 1, 11, and 12 spermatids, secondary spermatocytes and the Sertoli cells, and the meiotic figures showed an increase in the expression of p-p53ser315. An increase in the expression of a downstream target of p53 and protein 21cyclin-dependent kinase interacting protein 1/wild-type p53-activated factor 1 (p21CIP1/Waf1) in both diabetic groups did not show any time-dependent effects but occurred concurrent with an upregulation of p-p53ser315 in DM1 and a downregulation of the protein in DM3. In diabetic groups, the expression of p21CIP1/Waf1 was mainly cytoplasmic but also perinuclear in pachytene spermatocytes and round spermatids. The cytoplasmic localization of p21CIP1/Waf1 may be suggestive of an antiapoptotic role for the protein. The perinuclear localization is probably related to the cell cycle arrest meant for DNA damage repair. Diabetes upregulates p21CIP1/Waf1 signaling in testicular germ cells in association with alteration in p-p53ser315 expression, probably to counteract DNA damage-induced cell death.

Mitogen-activated protein kinase signaling and its association with oxidative stress and apoptosis in lead-exposed hepatocytes

Lead toxicity has become a serious public health concern all over the world. Previous studies have shown that lead induces biochemical and structural changes in liver. However, although lead is known to alter liver functions, the underlying molecular mechanisms of hepatotoxicity are not yet clear. We hypothesized that a correlation exists between oxidative stress, apoptosis and mitogen‐activated protein kinases (MAPKs) in lead‐exposed liver. Wistar rats were treated with 0, 0.5%, and 1% lead acetate for 3d, 14d, and 35d and sacrificed the next day. On 4d, oxidative stress and apoptosis were correlated with downregulated expressions of ERK1/2 and p38‐MAPKα/β, and upregulated expressions of JNK1/3 in males. In females, the correlation was with downregulated expressions of ERK1/2 and upregulated expressions of p38‐MAPKα/β and JNK1/3. On 15d, the correlation was observed with upregulated expressions of p38‐MAPKα/β in males and downregulated expressions of p38‐MAPKα/β in females. In both sexes, a correlation was observed with upregulated expressions of ERK1/2 and JNK1/3 in 1% groups. On 36d, the correlation was observed with downregulated expressions of p38‐MAPKα/β in males and their upregulated expressions in females. Time‐dependent increase in lipid peroxidation on 15d and 36d correlated with upregulated expressions of p38‐MAPKα/β in females and ERK1/2 in 1% groups in both sexes. The lower dose induced more apoptosis up to 15d in females and the higher dose induced in males on 36d. Generally, the female livers had more p38‐MAPKα/β than the male livers. On 36d, the female livers showed more p38‐MAPKα/β and JNK1/3 than the male livers. In conclusion, although not clearly defined, a correlation exists among oxidative stress, apoptosis, and the MAPKs in lead‐exposed hepatocytes. The sex‐dependent effects may be due to differences in hormonal or other physiological mechanisms. In lead‐exposed hepatocytes, the apoptosis may be induced via oxidative stress‐mediated alterations in the MAPKs.

Effects of bleomycin, etoposide and cisplatin treatment on Leydig cell structure and transcription of steroidogenic enzymes in rat testis

Cytotoxic anticancer chemotherapy affects pituitary-testicular hormonal axis in humans and in animals. This study investigated the effects on Leydig cells of three cycles of bleomycin, etoposide and cisplatin (0.75, 7.5, and 1.5mg/kg, respectively; BEP) chemotherapy in rat testis. The chemotherapy has induced hyperplasia of and degenerative changes in Leydig cells at the end of BEP exposure, which remained so even after a recovery time of 63 days. The increased testicular oxidative stress at the end of the chemotherapy returned to normal level after the recovery time. The chemotherapy has stimulated the transcription of scavenger receptor class type-B1 (SCARB1), steroidogenic acute-regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1, and inhibited that of 17β-hydroxysteroid dehydrogenase (HSD17B6) and CYP19A1 in association with increased cholesterol and decreased testosterone levels. Even after the recovery time, the chemotherapy still had inhibitory effects on the transcription of all of the above genes in addition to luteinizing hormone receptor and HSD3B1, but not on the StAR gene. The cholesterol and testosterone levels also did not show any significant differences with the control group. The decreased testosterone level at the end of chemotherapy was probably due to inhibition of HSD3B1 and HSD17B6 genes. In conclusion, clinically relevant dose-levels and treatment protocols of BEP chemotherapy adversely affect Leydig cell function. The BEP chemotherapy inhibits the transcription of steroidogenic enzymes and that these effects sustain over an extended period of time without returning to normal levels.

Expression and Subcellular Localization of Metastasis-Associated Protein 1, Its Short Form, and Estrogen Receptors in Rat Placenta:

Metastasis-associated protein 1 (MTA1) and its short form (MTA1s) regulate the function of estrogen receptors (ERs). Estrogens, via ERs, affect placental growth and fetal development, a process that may involve MTA1 signaling. Expression of MTA1, MTA1s, ERα, and ERβ genes and proteins in rat placentas was studied on 16, 19, and 21 days of gestation (dg). The ERβ messenger RNA decreased significantly toward the end of gestation, whereas its protein level increased in the nuclear fraction on 21 dg. Both MTA1 and MTA1s increased with gestation. Decidual, trophoblast giant, glycogen, and villous trophoblast cells expressed MTA1, ERα, and ERβ proteins on all dg with colocalization of MTA1 with ERα and ERβ in the nucleus and cytoplasm. Expression of MTA1 suggests a possible role in regulating placental functions; considering the repressive function of MTA1 on ERs, the expression of MTA1 suggests that placental cells may be less sensitive to estrogens during late pregnancy.

Trans-resveratrol mitigates type 1 diabetes-induced oxidative DNA damage and accumulation of advanced glycation end products in glomeruli and tubules of rat kidneys

&NA; Hyperglycemia induces the formation of advanced glycation end products (AGEs) and their receptors (RAGEs), which alter several intracellular signaling mechanisms leading to the onset and progression of diabetic nephropathy. The present study focused on, i) modulatory effects of trans‐resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene) on structural changes, AGE (N&egr;‐carboxymethyl‐lysine), RAGE, oxidative stress and DNA damage, and apoptosis, and ii) localization of fibrotic changes, AGE, RAGE, 8‐oxo‐dG and 4‐hydroxynonenal (4‐HNE) in diabetic rat kidneys. Resveratrol (5 mg/kg; po, administered during last 45 days of 90‐day‐long hyperglycemic period) administration to streptozotocin‐induced type 1 diabetic male Wistar rats reduced renal hypertrophy and structural changes (tubular atrophy, mesangial expansion or shrinkage, diffuse glomerulonephritis, and fibrosis), AGE accumulation, oxidative stress and DNA damage (8‐oxo‐dG), 4‐HNE, caspase‐3, and cleaved‐caspase‐3, but not the RAGE expression. The AGE accumulated in the mesangium, vascular endothelium, and proximal convoluted tubules and less intensely in distal convoluted tubules of diabetic rat kidneys. The RAGE expression increased in the convoluted tubules and collecting ducts of diabetic rat kidneys, but not in the mesangium. Diabetes increased the expression of 8‐oxo‐dG in nuclei and cytoplasm of renal cells, and 4‐HNE in glomeruli, convoluted tubules, the loops of Henle and collecting ducts. Hyperglycemia‐induced AGE‐RAGE axis and oxidative stress in turn induced apoptosis in diabetic kidneys. Resveratrol mitigated all diabetic effects except the RAGE expression. In conclusion, Resveratrol significantly alleviates diabetes‐induced glycation, oxidative damage, and apoptosis to inhibit the progression of diabetic nephropathy. Resveratrol supplementation may be useful to hinder the onset and progression of diabetic kidney diseases. Graphical abstract Figure. No caption available. HighlightsResveratrol inhibits diabetes‐induced histopathology and fibrosis in kidneys.Resveratrol mitigates AGE accumulation in major renal components.Resveratrol does not alleviate diabetes‐induced RAGE expression in kidneys.Resveratrol inhibits diabetes‐induced renal oxidative DNA damage and apoptosis.Resveratrol protects kidneys from diabetic glomerulonephritis and nephropathy.

Effects of antioxidants on drugs used against testicular cancer-induced alterations in metastasis-associated protein 1 signaling in the rat testis.

Metastasis-associated protein 1 (MTA1) is involved in tumor growth and metastasis of cancers. Being a component of nucleosome remodeling and histone deacetylase complex, the protein is also associated with DNA damage response pathway. Since the protein is involved in cancer pathology, we first investigated the effects of bleomycin, etoposide, and cisplatin (BEP) on MTA1 signaling in the testis. Second, since the antioxidants (AOs) have protective effects, we further investigated whether or not an AO cocktail modulates the effects of the drugs. Adult male Sprague Dawley rats (N = 4) were treated either with saline, or AO (α-tocopherol, l-ascorbic acid, zinc, and selenium), or therapeutic dose levels of etoposide (15 mg/kg) and cisplatin (3 mg/kg) from day 1–4 of the week and B (1.5 mg/kg) on the second day of the week, or BEP + AO. The real-time polymerase chain reaction showed that MTA1 and MTA1s (short form) gene expression was downregulated in AO (100% and 100%), BEP (86% and 71%), and BEP + AO (97% and 93%) groups. Western blotting and immunohistochemistry results showed that unnormalized MTA1 protein expression was upregulated in AO (38%) and BEP + AO (34%) groups; however, the MTA1/β-actin ratio was upregulated in all treated groups (21, 19, and 15%, respectively). In conclusion, the results indicate that both BEP and AO suppress MTA1 and MTA1s transcription, which may render the germ cells to be more prone to apoptosis. However, upregulation of MTA1 protein expression may be related to induced DNA damage. Modulation of MTA1 signaling is a novel mechanism of action of BEP and AO, which may be useful in developing newer anticancer drugs.

Effects of diabetes on retinal pigment epithelial cell proliferation and mitogen-activated protein kinase signaling in dark Agouti rats

Although diabetes induces retinopathy its effects on retinal pigment epithelium (RPE) are not clearly known. The present study investigated the effects of streptozotocin-induced diabetes on RPE cell proliferation and the expression of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinases (JNKs) in rats. The bromodeoxyuridine immunohistochemistry revealed diabetes induced RPE cell proliferation at the end of first and second weeks in dark Agouti rats and at the end of first week in Wistar rats, but it inhibited the proliferation in both strains at the end of fifth week (P<0.05). A further analysis at the end of second week in the dark Agouti rats showed the cell proliferation, but not apoptosis, in association with an increase in ERK1/2 expression (P<0.05). However, the increased ERK level did not affect the expression of one of its substrates, the transcription factor c-Fos, suggesting that this protein has no role in the induction of the RPE cell proliferation. On the other hand, although total JNKs showed a decrease in the diabetic group (P<0.05), the JNKp46 isoform was increased and the JNKp54 isoform was decreased, but without any effects on one of their substrates, the transcription factor, c-Myc. Our results indicate that the RPE cell proliferation in diabetic rats may be mediated through mitogen-activated protein kinases. Thus, modulation of mitogen-activated protein kinases signaling may be a putative therapeutic option to alleviate the genesis of diabetes-induced retinal disruptions including retinopathy.