Effect of melatonin and time of administration on irradiation-induced damage to rat testes

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

Comparison of I-FISH and G-banding for the detection of chromosomal abnormalities during the evolution of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) patients with a normal karyotype constitute a heterogeneous group from a biological standpoint and their outcome is often unpredictable. Interphase fluorescence in situ hybridization (I-FISH) studies could increase the rate of detection of abnormalities, but previous reports in the literature have been contradictory. We performed I-FISH and conventional karyotyping (G-banding) on 50 MDS patients at diagnosis, after 6 and 12 months or at any time if a transformation to acute myeloid leukemia (AML) was detected. Applying a probe-panel targeting the centromere of chromosomes 7 and 8, 5q31, 5p15.2 and 7q31, we observed one case with 5q deletion not identified by G-banding. I-FISH at 6 and 12 months confirmed the karyotype results. Eight cases transformed to AML during follow-up, but no hidden clone was detected by I-FISH in any of them. The inclusion of I-FISH during follow-up of MDS resulted in a small improvement in abnormality detection when compared with conventional G-banding.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Glomerular damage as a predictor of renal allograft loss

Interstitial fibrosis and tubular atrophy (IF/TA) are the most common cause of renal graft failure. Chronic transplant glomerulopathy (CTG) is present in approximately 1.5-3.0% of all renal grafts. We retrospectively studied the contribution of CTG and recurrent post-transplant glomerulopathies (RGN) to graft loss. We analyzed 123 patients with chronic renal allograft dysfunction and divided them into three groups: CTG (N = 37), RGN (N = 21), and IF/TA (N = 65). Demographic data were analyzed and the variables related to graft function identified by statistical methods. CTG had a significantly lower allograft survival than IF/TA. In a multivariate analysis, protective factors for allograft outcomes were: use of angiotensin-converting enzyme inhibitor (ACEI; hazard ratio (HR) = 0.12, P = 0.001), mycophenolate mofetil (MMF; HR = 0.17, P = 0.026), hepatitis C virus (HR = 7.29, P = 0.003), delayed graft function (HR = 5.32, P = 0.016), serum creatinine ≥1.5 mg/dL at the 1st year post-transplant (HR = 0.20, P = 0.011), and proteinuria ≥0.5 g/24 h at the 1st year post-transplant (HR = 0.14, P = 0.004). The presence of glomerular damage is a risk factor for allograft loss (HR = 4.55, P = 0.015). The presence of some degree of chronic glomerular damage in addition to the diagnosis of IF/TA was the most important risk factor associated with allograft loss since it could indicate chronic active antibody-mediated rejection. ACEI and MMF were associated with better outcomes, indicating that they might improve graft survival.

Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm², pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Decreased levels of pNR1 S897 protein in the cortex of neonatal Sprague Dawley rats with hypoxic-ischemic or NMDA-induced brain damage

Our objective was to investigate the protein level of phosphorylated N-methyl-D-aspartate (NMDA) receptor-1 at serine 897 (pNR1 S897) in both NMDA-induced brain damage and hypoxic-ischemic brain damage (HIBD), and to obtain further evidence that HIBD in the cortex is related to NMDA toxicity due to a change of the pNR1 S897 protein level. At postnatal day 7, male and female Sprague Dawley rats (13.12 ± 0.34 g) were randomly divided into normal control, phosphate-buffered saline (PBS) cerebral microinjection, HIBD, and NMDA cerebral microinjection groups. Immunofluorescence and Western blot (N = 10 rats per group) were used to examine the protein level of pNR1 S897. Immunofluorescence showed that control and PBS groups exhibited significant neuronal cytoplasmic staining for pNR1 S897 in the cortex. Both HIBD and NMDA-induced brain damage markedly decreased pNR1 S897 staining in the ipsilateral cortex, but not in the contralateral cortex. Western blot analysis showed that at 2 and 24 h after HIBD, the protein level of pNR1 S897 was not affected in the contralateral cortex (P > 0.05), whereas it was reduced in the ipsilateral cortex (P < 0.05). At 2 h after NMDA injection, the protein level of pNR1 S897 in the contralateral cortex was also not affected (P > 0.05). The levels in the ipsilateral cortex were decreased, but the change was not significant (P > 0.05). The similar reduction in the protein level of pNR1 S897 following both HIBD and NMDA-induced brain damage suggests that HIBD is to some extent related to NMDA toxicity possibly through NR1 phosphorylation of serine 897.

Role of p53 codon 72 polymorphism in chromosomal aberrations and mitotic index in patients with chronic hepatitis B

Polymorphisms of the p53 gene, which participates in DNA repair, can affect the functioning of the p53 protein. The Arg and Pro variants in p53 codon 72 were shown to have different regulation properties of p53-dependent DNA repair target genes that can affect various levels of cytogenetic aberrations in chronic hepatitis B patients. The present study aimed to examine the frequency of chromosomal aberrations and the mitotic index in patients with chronic hepatitis B and their possible association with p53 gene exon 4 codon 72 Arg72Pro (Ex4+119 G>C; rs1042522) polymorphism. Fifty-eight patients with chronic hepatitis B and 30 healthy individuals were genotyped in terms of the p53 gene codon 72 Arg72Pro polymorphism by PCR-RFLP. A 72-h cell culture was performed on the same individuals and evaluated in terms of chromosomal aberrations and mitotic index. A high frequency of chromosomal aberrations and low mitotic index were detected in the patient group compared to the control group. A higher frequency of chromosomal aberrations was detected in both the patient and the control groups with a homozygous proline genotype (13 patients, 3 control subjects) compared to patients and controls with other genotypes [Arg/Pro (38 patients, 20 control subjects) and Arg/Arg (7 patients, 7 control subjects)]. We observed an increased frequency of cytogenetic aberrations in patients with chronic hepatitis B. In addition, a higher frequency of cytogenetic aberrations was observed in p53 variants having the homozygous proline genotype compared to variants having other genotypes both in patients and healthy individuals.

Evaluation of chromosomal abnormalities by cIg-FISH and association with proliferative and apoptotic indexes in multiple myeloma

Eighty-six newly diagnosed multiple myeloma (MM) patients from a public hospital of São Paulo (Brazil) were evaluated by cIg-FISH for the presence of del(13)(q14), t(4;14)(p16.3;q32) and del(17)(p13). These abnormalities were observed in 46.5, 9.3, and 7.0% of the patients, respectively. In order to identify the possible role of del(13)(q14) in the physiopathology of MM, we investigated the association between this abnormality and the proliferative and apoptotic indexes of plasma cells. When cases demonstrating t(4;14)(p16.3;q32) and del(17)(p13) were excluded from the analysis, we observed a trend towards a positive correlation between the proportion of cells carrying del(13)(q14) and plasma cell proliferation, determined by Ki-67 expression (r = 0.23, P = 0.06). On the other hand, no correlation between the proportion of cells carrying del(13)(q14) and apoptosis, determined by annexin-V staining, was detected (r = 0.05, P = 0.69). In general, patients carrying del(13)(q14) did not have lower survival than patients without del(13)(q14) (P = 0.15), but patients with more than 80% of cells carrying del(13)(q14) showed a lower overall survival (P = 0.033). These results suggest that, when del(13)(q14) is observed in a high proportion of malignant cells, it may have a role in determining MM prognosis. Another finding was a statistically significant lower overall survival of patients with t(4;14)(p16.3;q32) (P = 0.026). In the present study, almost half the patients with t(4;14)(p16.3;q32) died just after diagnosis, before starting treatment. This fact suggests that, in São Paulo, there may be even more patients with this chromosomal abnormality, but they probably die before being diagnosed due to unfavorable socioeconomic conditions. This could explain the low prevalence of this chromosomal abnormality observed in the present study.

Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. This study developed a model that can mimic the intestinal heat stress environment. It demonstrates the effects on intestinal epithelial cell damage, and indicated that propofol could be used as a therapeutic drug for the treatment of heat-stress-induced intestinal injuries.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg,ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

Lipid peroxidation, detoxification capacity, and genome damage in mice after transplacental exposure to pharmaceutical drugs

Data on genome damage, lipid peroxidation, and levels of glutathione peroxidase (GPX) in newborns after transplacental exposure to xenobiotics are rare and insufficient for risk assessment. The aim of the current study was to analyze, in an animal model, transplacental genotoxicity, lipid peroxidation, and detoxification disturbances caused by the following drugs commonly prescribed to pregnant women: paracetamol, fluconazole, 5-nitrofurantoin, and sodium valproate. Genome damage in dams and their newborn pups transplacentally exposed to these drugs was investigated using the in vivo micronucleus (MN) assay. The drugs were administered to dams intraperitoneally in three consecutive daily doses between days 12 and 14 of pregnancy. The results were correlated, with detoxification capacity of the newborn pups measured by the levels of GPX in blood and lipid peroxidation in liver measured by malondialdehyde (HPLC-MDA) levels. Sodium valproate and 5-nitrofurantoin significantly increased MN frequency in pregnant dams. A significant increase in the MN frequency of newborn pups was detected for all drugs tested. This paper also provides reference levels of MDA in newborn pups, according to which all drugs tested significantly lowered MDA levels of newborn pups, while blood GPX activity dropped significantly only after exposure to paracetamol. The GPX reduction reflected systemic oxidative stress, which is known to occur with paracetamol treatment. The reduction of MDA in the liver is suggested to be an unspecific metabolic reaction to the drugs that express cytotoxic, in particular hepatotoxic, effects associated with oxidative stress and lipid peroxidation.

Leukocytosis, muscle damage and increased lymphocyte proliferative response after an adventure sprint race

The effect of an adventure sprint race (ASR) on T-cell proliferation, leukocyte count and muscle damage was evaluated. Seven young male runners completed an ASR in the region of Serra do Espinhaço, Brazil. The race induced a strong leukocytosis (6.22±2.04×103 cells/mm3 beforevs 14.81±3.53×103 cells/mm3after the race), marked by a significant increase of neutrophils and monocytes (P<0.05), but not total lymphocytes, CD3+CD4+ or CD3+CD8+ cells. However, the T-cell proliferative response to mitogenic stimulation was increased (P=0.025) after the race, which contradicted our hypothesis that ASR, as a high-demand competition, would inhibit T-cell proliferation. A positive correlation (P=0.03, r=0.79) was observed between the proliferative response of lymphocytes after the race and the time to complete the race, suggesting that the proliferative response was dependent on exercise intensity. Muscle damage was evident after the race by increased serum levels of aspartate amino transferase (24.99±8.30 vs 50.61±15.76 U/L, P=0.003). The results suggest that humoral factors and substances released by damaged muscle may be responsible for lymphocyte activation, which may be involved in muscle recovery and repair.