DNA damage in lead-exposed hepatocytes: coexistence of apoptosis and necrosis?

The aim of the present study was to investigate the coexistence of oxidative DNA damage and apoptosis- and necrosis-related DNA damage, and to correlate with ultrastructural changes in hepatocyte nuclei in the lead-nitrate-exposed liver. Adult male Wistar rats were exposed to 0, 0.5, and 1% lead nitrate for 60 days, and the livers were sampled the next day. Ultrastructurally, hepatocyte nuclei showed no apoptosis-related morphological changes, but showed necrotic changes. Competitive enzyme-linked immunosorbent assay showed no change in 8-oxo-dG activity (P > 0.05), but immunohistochemistry showed its localization in hepatocytes, Kupffer cells, endothelium, and bile ductule epithelium. TUNEL-labeled DNA breaks presenting 3'-OH ends increased in hepatocytes in all functional zones of the portal acini and bile ductule epithelium (zones I>III>II). In situ oligo ligation revealed the existence of DNA breaks bearing duplex 3' overhangs and 5' P-blunt ends in hepatocytes of all functional zones and bile ductule epithelium. In conclusion, both apoptosis- and necrosis-related DNA damage coexist without significant oxidative DNA damage. Hepatocytes display changes related to necrosis, but not those related to apoptosis.

Molecular effects of chemotherapeutic drugs and their modulation by antioxidants in the testis.

Cisplatin-based chemotherapy regimens are preferred in the treatment of a variety of cancers. The present study investigated early cumulative molecular effects of therapeutic dose-levels of bleomycin, etoposide and cisplatin (BEP) in the testis and their modulation by an antioxidant cocktail (AO). Adult male Sprague-Dawley rats (N=7/group [G]) were treated with BEP as follows: G1 - control; G2 - AO (α-tocopherol [100 mg/kg], l-ascorbic acid [50 mg/kg], Zn [40 mg/l] and Se [100 µg/l]); G3 - B, 1.5 mg/kg on day 2; E, 15 mg/kg and P, 3 mg/kg for 4 days, and G4 - similar to G3 but also treated with AO for 4 days. In G3, the testis weight, sperm count and motility, and activities of enzymatic antioxidants decreased and lipid peroxidation increased compared to that in G1 (P<0.05). Seminiferous epithelial sloughing and degeneration were observed. In G3, mRNA levels of p53, Bcl-2 and Bax were unaltered but protein expression of p53 and Bax was up-regulated and that of Bcl-2 was down-regulated (P<0.05). These changes led to an increase in terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) positive germ cells indicating cell death (P<0.05). The AO recovered the BEP-induced molecular alterations to control levels. The mechanism of BEP-induced early testicular damage involves the initiation of oxidative stress, up-regulation of pro-apoptotic proteins and induction of cell death. Further, the induced testicular structural changes are negligible and less than those observed in single drug exposure studies reported in literature. The AO significantly ameliorates the BEP-induced pathogenesis of testicular damage suggesting its potential therapeutic uses.

Cisplatin induces duplex 3' overhangs and 5' blunt ends in epididymal epithelium in a Bax-dependent manner without any protection from L-ascorbic acid.

The ability of cisplatin (cis-diaminedichloroplatinum-II) to induce DNA double-strand breaks and expression of p53, Bax and Bcl-2 and possible protective effects of L-ascorbic acid was investigated in epididymis. Adult BALB/C mice were treated (ip) as follows: group I-- control; group II--L-ascorbic acid (10 mg/kg); group III--two cycles of 5 days each of cisplatin 1 mg/kg; group IV--cisplatin 1 mg/kg + L-ascorbic acid; group V--cisplatin 2.5 mg/kg; and group VI--cisplatin 2.5mg/kg + L-ascorbic acid. A recovery period of 17 days was given between the cycles. All animals were sacrificed within 72 h after the last treatment of cisplatin. The organ weight was decreased and the structural changes were observed. The tubular diameter and epithelial thickness were decreased in groups III to V, but recovered in groups IV and VI, except the epithelial thickness in group VI. The oxidative stress marker, 8-oxo-dG was expressed in groups III to VI without any protection by L-ascorbic acid. Intensive nuclear in situ oligo ligation (ISOL) and cytoplasmic Bax expression was observed in groups III-VI without any protective effects from L-ascorbic acid. p53 expression was reduced in groups III and V. Bcl-2 staining was weakly positive in all groups except groups III and V. In conclusion, cisplatin induces structural changes, oxidative stress, irreparable DNA double-strand breaks bearing duplex 3' dT overhangs and 5' P-blunt ends in a Bax-dependent manner, but L-ascorbic acid has little, if any, preventive effects on cisplatin-induced epididymal damage.

A purine nucleoside analogue-acyclovir [9-(2-hydroxyethoxymethyl)-9h-guanine] reversibly impairs testicular functions in mouse.

The present study was designed to investigate testicular effects of Acyclovir [9-(2-hydroxyethoxymethyl)-9H-guanine] in mouse. Swiss albino male mice (N=6/group/dose/sample time) were treated (i. p.) every day with 4, 16, 32, and 48 mg/kg body weight of Acyclovir for 15 d. The testis was examined for histopathological changes and the seminiferous tubular diameter (STD) and epithelial height (SE) were measured. The sperm count, sperm motility and sperm morphology assay in caudae epididymes, and intra-testicular levels lactate dehydrogenase (LDH) and testosterone were estimated on 7 d, 14 d, 21 d, 28 d, 35 d, and 70 d, following the last exposure. Acyclovir did not affect the body weight, but decreased the testis weight on 21 d and 28 d at two higher dose-levels, and on 35 d at all dose-levels. The STD was decreased on 21 d at 16-48 mg/kg dose-levels (p<0.01), on 28 d and 35 d, at all dose-levels (p<0.01-0.001). The SE was decreased on 14 d at two higher dose-levels (p<0.01), thereafter up to 35 d at all dose-levels (p<0.001). Acyclovir decreased the sperm count from 7-35 d (p<0.001) with a recovery observed on 70 d, except at 48 mg/kg dose-level (p<0.05), and inhibited the sperm motility (p<0.001) from 7-35 d with a maximum effect on 28-35 d. On the other hand, sperm abnormalities increased on 21 d, 28 d and 35 d at 16-48 mg/kg, at 32-48 mg/kg, and at 32 mg/kg dose-levels, respectively (P<0.05). LDH activity was increased (p<0.05-0.001) from 7 d (except at 4 mg/kg) to 35 d. Only 48 mg/kg dose group showed increase in LDH concentration on 70 d. In conclusion, Acyclovir is cytotoxic to germ cells inducing the cellular destruction, and reducing the sperm count and motility, and increasing sperm abnormalities. Further, Acyclovir also caused cellular destruction thus releasing LDH from the cells, and affecting the Leydig cell function. All adverse effects of Acycovir are reversible by 70 d, except the sperm count and LDH level, which appear to be affected over an extended period of time at a higher dose-level.

An aminoglycoside antibiotic gentamycin induces oxidative stress, reduces antioxidant reserve and impairs spermatogenesis in rats.

Gentamycin (GS) is an aminoglycoside antibiotic used to treat infections of various Gram-negative organisms. The present study was designed to investigate the effects of GS on oxidative stress, antioxidant levels, testicular structure and sperm parameters in the rat. Adult Wistar rats (12 week old; N=7/group) were treated (i. p.) with 0 mg/kg, 3 mg/kg and 5 mg/kg for 10 days at an interval of 24 hr between subsequent treatments. Animals were sacrificed on days 1 and 35 after the last treatment, and the reproductive organs were removed and weights of testis and seminal vesicle were recorded. The right testis was processed for light microscopic analysis. The left testis was homogenized and step 19 spermatids were counted to determine the daily sperm production (DSP) and daily abnormal sperm production (DASP). The sperm count, sperm motility and incidence of abnormal sperms were estimated in the epididymis. In testicular sections, along with the evaluation of qualitative changes, the seminiferous tubule diameter (STD) and the epithelial height (SE) were measured. The incidence of stage XIV tubules in testicular sections, meiotic figures and step 14 spermatids/stage XIV tubule, and step 19 spermatids/stage VII tubule were estimated. Intra-testicular levels of superoxide anion, lipid peroxidation and antioxidants-superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and ascorbic acid were measured. GS did not affect the body weight, but the testis weight and DSP were decreased at 5 mg/kg dose-level on both days (p<0.05), and the weight of seminal vesicle decreased on day 35 at both dose-levels. The DASP was increased in a dose-dependent manner (p<0.05) on days 1 and 35 at both dose-levels. The sperm count was decreased at both dose-levels on day 35, whereas the sperm motility was decreased and sperm abnormality was increased on day 1 at 5 mg/kg and on day 35 at both dose-levels. GS induced structural changes such as sloughing of seminiferous epithelium, vacuoles and gaps in the epithelium, nuclear pyknosis and atrophic changes in a few tubules. The tubular shrinkage was observed as indicated by decreased STD and SE on both days at 5 mg/kg dose-level. Incidence of stage XIV tubules and step 19 spermatids/stage VII tubule decreased on all time points at all dose-levels, whereas the step 14 spermatids and meiotic figures decreased on day 35 at both dose-levels (p<0.05). The free radical- superoxide anion concentration was significantly increased on day 1 in a dose-dependent pattern (p<0.05). However, activities of all 3 enzymatic antioxidants and ascorbic acid level decreased in a dose-dependent pattern on day 1 (p<0.05), except the GPx, which was also decreased on day 35 at 5 mg/kg dose-level. There was a significant rise in the thiobarbituric acid reactive substances on day 1 indicating increased lipid peroxidation in the testis. In conclusion, GS induces an oxidative stress-status in the testis by increasing free radical formation and lipid peroxidation, and by decreasing the antioxidant reserves. These biochemical changes manifest as structural and cytotoxic changes in the testis. Further, GS also affects the spermatozoa by affecting their number, motility and morphology.

An organophosphate insecticide methyl parathion (o- o- dimethyl o-4-nitrophenyl phosphorothioate) induces cytotoxic damage and tubular atrophy in the testis despite elevated testosterone level in the rat.

Methyl parathion (MP) is an organophosphate pesticide used in agriculture, although quite often illegally used indoors to contain insects. The present study was planned to investigate the effects of MP on rat testis. Adult male Wistar rats (13-14 weeks) were treated with MP as follows. Experiment 1-0, 1.75, 3.5 or 7 mg/kg i.p. for 5 days and sacrificed on Day 14; experiment 2 and 3- 0, 0.5, or 1 mg/kg i.p. for 12 days, and sacrificed on Days 130 and 77, respectively; experiment 4- 0, 0.75, or 1.5 mg/kg i.p. for 25 days, and sacrificed on Day 17; experiment 5- 0 or 3.5 mg/kg po for 25 days, and sacrificed on Day 17, after the last exposure. MP decreased the body weight and the testis weight in experiments 4 and 5 (p<0.05-0.001) due to decreased food intake and tubular atrophy respectively. MP increased the intra-testicular testosterone level and decreased the LH level in experiments 4 and 5. The seminiferous epithelium showed sloughing of germ cells, vacuoles, focal necrosis, and formation of multinucleated giant cells, cellular degeneration (nuclear pyknosis, halo appearance and shrinkage of nuclei) and tubular atrophy, especially in experiment 4. The degree of testicular damage was higher in experiment 4>5>1>3>2 indicating more effect of prolonged i.p. treatment. Homogenization-resistant spermatid count was decreased in experiments 1, 4 and 5, and MP also decreased the tubular diameter, and epithelial height (p<0.05-0.001). Incidences of stage XIV tubules, number of meiotic figures and elongating spermatids were also decreased, whereas the incidence of tubules showing epithelial sloughing increased (p<0.05-0.001). We conclude that MP is a reproductive toxicant in male rats which causes significant testicular damage in the testis.

Occurrence and fate of palmar type of median artery.

BACKGROUND: The median artery represents a persistent part of the embryonic arterial axis of the upper extremity. It appears mainly as two types: an antebrachial type and a palmar type. The palmar type is of major clinical significance. METHOD: This study was undertaken to investigate the incidence and fate of the palmar type of the median artery in 19 cadavers. The occurrence was 15.8% and of this two incidences (5.2%) were on the right side and four (10.6%) were on the left side. On the right side, the artery originated from the ulnar and joined with the superficial palmar arch or anterior interosseous artery and communicated with the radial artery. CONCLUSIONS: This study concludes that palmar type of median artery is found at a higher incidence than the antebrachial type and that it may be involved in the pronator teres syndrome, carpal tunnel syndrome and anterior interosseous syndrome.

The incidence of a superficial arterial pattern in the human upper extremities.

The incidence of superficial arteries was studied in 68 (38 right and 30 left) upper extremities. One right limb of an adult male presented a superficial arterial pattern (2.63%, total 1.47%) resembling a superficial brachio-ulno-radial artery (SBUR). The median nerve crossed the superficial brachial artery (SBA) from the posterior to the medial side and again posterior to the same at the cubital fossa. The superficial brachial artery divided into superficial radial and superficial ulnar arteries, which coursed distally superficial to the muscles but deep to the deep fascia. The superficial radial artery passed deep to the extensor tendons of the thumb. The superficial ulnar artery gave only muscular branches in the forearm. The superficial radial artery gave origin to the radial recurrent artery and the common interosseous trunk. The latter gave origin to a palmar type of median artery, muscular branches, and an artery that divided into anterior and posterior ulnar recurrent arteries. It also gave origin to the anterior and posterior interosseous arteries. The latter provided the interosseous recurrent artery and a branch that coursed towards the olecranon process of the ulna. The knowledge of this variation is important since it may be compromised in surgical procedures of the upper limb.