Regeneration and change of muscle fiber types after injury induced by a hemorrhagic fraction isolated from Agkistrodon contortrix laticinctus venom.

Tibialis anterior (TA) muscles of rats were evaluated 3h, 3 and 30days after intramuscular injection of ACL hemorrhagic toxin I (ACLHT-I, 5mg/kg), partially purified from the venom of Agkistrodon contortrix laticinctus. Contralateral muscles were injected with saline. Three hours after ACLHT-1 injection: presence of hemorrhagic areas and myonecrotic muscle fibers. Three days: injured muscles showed areas in regeneration, some regions with delay of regeneration and bundles of normal fibers. An increased TA muscle weight was found when compared with the contralateral (0.45+/-0.03g versus 0.36+/-0.04g, p=0.04). Thirty days: areas of regenerated muscle fibers presented splits and centralized nuclei. Some regions were replaced by connective tissue. All muscle fiber types were injured but only the incidence of type IIC increased (3.4+/-2.0% versus 0.2+/-0.2%, p=0.0005). Regenerated areas of muscles were exclusively composed by fiber types II and IIC. Regenerated muscles decreased the muscle weight (0.49+/-0.1g versus 0.66+/-0.05g, p=0. 03). In conclusion, ACLHT-I: (a) caused hemorrhage and muscle fiber injury; (b) injured both fiber types I and II; (c) increased the incidence of fiber type IIC and; (d) some muscle regions were replaced by connective tissue.     

Systemic skeletal muscle necrosis induced by crotoxin.

Systemic skeletal muscle necrosis induced by crotoxin, the major component of the venom of Crotalus durissus terrificus, was investigated. Mice received an intramuscular injection of crotoxin (0.35mg/kg body weight) into the right tibialis anterior (TA) muscles, which were evaluated 3h, 24h and 3 days later. Control mice were injected with saline. Right and left TAs, gastrocnemius, soleus and right masseter and longissimus dorsi were removed and frozen. Histological sections were stained with Toluidine Blue or incubated for acidic phosphatase reaction. Three and 24h after the injection, signals of muscle fiber injury were found: (a) in the injected TA muscles; (b) in both right and contralateral soleus and red gastrocnemius; and (c) in the masseter muscles. Contralateral TA, longissimus dorsi and white gastrocnemius muscles were not injured. In conclusion, crotoxin induced a systemic and selective muscle injury in muscles or muscle regions composed by oxidative muscle fibers.     

Investigation of antioxidant,anti-inflammatory and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats

The present study investigated the preventive effect of eugenol, a naturally occurring food flavouring agent on thioacetamide (TA)-induced hepatic injury in rats. Adult male Wistar rats of body weight 150–180 g were used for the study. Eugenol (10.7 mg/kg b.w./day) was administered to rats by oral intubation for 15 days. TA was administered (300 mg/kg b.w., i.p.) for the last 2 days at 24 h interval and the rats were sacrificed on the 16th day. Markers of liver injury (aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase and bilirubin), inflammation (myeloperoxidase, tumor necrosis factor-α and interleukin-6), oxidative stress (lipid peroxidation indices, protein carbonyl and antioxidant status) and cytochrome P4502E1 activity were assessed. Expression of cyclooxygenase-2 (COX-2) and the extent of DNA damage were analyzed using immunoblotting and comet assay, respectively. Liver injury and collagen accumulation were assessed using histological studies by hematoxylin and eosin and Masson trichrome staining. Rats exposed to TA alone showed increased activities of hepatocellular enzymes in plasma, lipid peroxidation indices, inflammatory markers and pro-inflammatory cytokines and decreased antioxidant status in circulation and liver. Hepatic injury and necrosis were also evidenced by histology. Eugenol pretreatment prevented liver injury by decreasing CYP2E1 activity, lipid peroxidation indices, protein oxidation and inflammatory markers and by improving the antioxidant status. Single-cell gel electrophoresis revealed that eugenol pretreatment prevented DNA strand break induced by TA. Increased expression of COX-2 gene induced by TA was also abolished by eugenol. These findings suggest that eugenol curtails the toxic effects of TA in liver.     

Satellite cell activity in muscle regeneration after contusion in rats

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time‐course of satellite cell activity (from 3 h to 7 days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real‐time polymerase chain reaction (PCR). 2. Inflammation of the injured muscle occurred within 6 h, followed by disintegration of the damaged myofibres within 12 h. Newly formed myofibres appeared by Day 7. 3. The number of MyoD‐positive nuclei (activated satellite cells) in the injured muscle was significantly increased by 6 h, reaching a maximum by 12 h after contusion. However, the number of MyoD‐positive nuclei decreased towards control levels by Day 7. Changes in the number of bromodeoxyuridine‐labelled nuclei (proliferating satellite cells) paralleled the changes seen in the number of MyoD‐positive nuclei. Conversely, expression of myogenin protein was not apparent until Day 3 and increased further by Day 7. Colabelling of MyoD and myogenin was seen in only a few cells. 4. The time‐course of MyoD mRNA expression corresponded with MyoD protein expression. However, there were two peaks in myogenin mRNA expression: 6 h and Day 7 after contusion. The second peak coincided with upregulation of myostatin mRNA levels. 5. The results of the present study suggest that contusion activates a homogeneous population of satellite cells to proliferate within 3 days, followed by differentiation to form new myofibres. The latter may be regulated, in part, by myostatin.     

Effect of low-voltage electrical stimulation on angiogenic growth factors in ischaemic rat skeletal muscle

1. Low-voltage electrical stimulation (LVES) in skeletal muscle at a level far below the threshold of muscle contraction has been reported to promote local angiogenesis. However, the mechanism underlying the promotion of local angiogenesis by LVES has not been fully elucidated. In the present study, we evaluated whether angiogenic factors, such as vascular endotherial growth factor (VEGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), as well as other disadvantageous factors, such as inflammation (interleukin (IL)-6) and hypoxia (hypoxia-inducible factor (HIF)-1alpha), contribute to the local angiogenesis produced by LVES. 2. We completely excised bilateral femoral arteries of male Sprague-Dawley rats. After the operation, electrodes were implanted onto the centre of the fascia of the bilateral tibialis anterior (TA) muscles, tunnelled subcutaneously and exteriorized at the level of the scapulae. The right TA muscles of rats were stimulated continuously at a stimulus frequency of 50 Hz, with a 0.1 V stimulus strength and no interval, for 5 days. The left TA muscles served as controls. 3. We found that both VEGF and HGF protein were significantly increased by LVES in stimulated muscles compared with control. The VEGF level of the LVES group was 89.10 +/- 17.19 ng/g, whereas that of the control group was 65.07 +/- 12.88 ng/g, as determined by ELISA (P < 0.05). The HGF level of the LVES and control groups was 8.52 +/- 1.96 and 5.80 +/- 2.14 ng/g, respectively (P < 0.05). In contrast, there was no difference in FGF, IL-6 and HIF-1alpha between the LVES and control groups. 4. These results suggest that LVES in a hindlimb ischaemia model of rats increases not only VEGF, but also HGF, production, which may be the main mechanism responsible for the angiogenesis produced by LVES.     

Effects of low concentrations of benzene on human lung cells in vitro

Exposure to benzene causes health hazards to humans. The airway epithelium is a physical barrier to inhaled toxicants and particulates. This is an in vitro basic science study to evaluate the effects of benzene on lung cells without the inflammatory responses triggered by inhalation. Dose–response cytotoxicity was assessed using two cell lines: alveolar derived (A549) human epithelial adenocarcinoma and human lung (LL24) fibroblast. A549 cells were more resistant than LL24 fibroblast lung cells to benzene. LL24 cells demonstrated enhanced proliferation with diluted benzene solutions. Moreover, low concentrations of benzene enhanced telomerase activity in LL24 cells while no effects were observed in the adenocarcinoma cells. Proteolysis of lung matrix by matrix metalloproteinases (MMPs) is an early event observed in lung pathologies. Benzene increased MMP-2 and MMP-3 mRNA. Using the ratio (MMP-1 + MMP-2 + MMP-3)/(TIMP-1 + TIMP-2), as an index of prodestructive activity, we observed a dose-dependent increase. The overall higher expression levels of MMPs in benzene treated cells did not appear to be controlled by TIMPs, which are negatively correlated.     

Non-coplanar 2,2′,3,3′,4,4′,5,5′,6,6′-decachlorobiphenyl (PCB 209) did not induce cytochrome P450 enzyme activities in primary cultured rat hepatocytes,was not genotoxic,and did not exhibit endocrine-modulating activities

2,2′,3,3′,4,4′,5,5′,6,6′-Decachlorobiphenyl (PCB 209) is a fully chlorinated, non-coplanar biphenyl. To demonstrate that PCB 209 is not likely to exhibit human health hazards common to coplanar PCBs it was tested for cytochrome P450 (P450) enzyme induction potentials, genetic toxicity, and endocrine-modulating activity. PCB 209 (dose from 0.005 to 5000 ng/mL) did not significantly induce P450 CYP1A, 2A, 2B, 3A, or 4A enzyme activities in primary cultured rat hepatocytes. In contrast, Aroclor 1260, a PCB mixture that contains approximately 60% chlorine by weight, showed significant induction of P450 CYP1A, 2A, 2B, and 3A within the same dose range. PCB 209 (dose from 100 to 5000 μg/plate) was negative in the bacterial mutagenicity (Ames) test in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 or in Eschericia coli strain WP2uvrA. PCB 209 (dose from 25 to 150 μg/mL) was also negative for forward mutations at the thymidine kinase (TK^+/−) locus of L5178Y mouse lymphoma cells. The Ames and the mouse lymphoma assays were both conducted in the absence and presence of rat liver S9 fraction. PCB 209 (dose from 500 to 2000 mg/kg by single dose oral gavage) did not induce an increase in the frequency of micronuclei in polychromatic erythrocytes in mouse bone marrow in vivo. PCB 209 did not induce estrogenic effects when administered by gavage to ovariectomized adult female rats at 500 and 1000 mg/kg for 4 days, nor did it produce alterations consistent with endocrine-modulating activity in adult intact male rats when administered by gavage at 500 and 1000 mg/kg for 15 consecutive days.     

Ethylacetate fraction from Korean seaside starfish, Asterias amurensis, has an inhibitory effect on MMP-9 activity and expression and on migration behavior of TNF-α induced human aortic smooth muscle cells

Atherosclerosis is accompanied by the proliferation of human aortic smooth muscle cells (HASMC) and their movement into the intima. Many reports have indicated the involvement of gelatinases (MMP-9 and MMP-2) in this pathogenesis. The ethylacetate fraction from starfish, Asterias amurensis (EFA), harvested from the Korean seaside has an inhibitory effect on MMP-9 and MMP-2 activities, as well as on the expression of MMP-9 in TNF-α induced HASMC in a dose-dependent manner. Also, EFA inhibits the migration of TNF-α induced HASMC in transwells containing gelatin coated plugs. EFA was not cytotoxic to HASMC over the range 0-1 mg/ml. By Western-blot analysis, it was revealed that the phosphorylation of extracellular signal regulated kinase (ERK) in TNF-α induced cells was inhibited and nuclear factor kappa B (NF-κB) p65 levels in nuclear extracts were decreased by EFA treatment. In addition, ERK inhibitor (U0126) treated cells exhibited decreased MMP-9 activity in the zymographic assay. From these results, it was found that the gelatinolytic activity was regulated (1) by enzymatic inhibition of both MMP-9 and MMP-2, as well as (2) by the decreased production of MMP-9 via ERK pathways in EFA treated HASMCs. Taken together, it has been shown that EFA has a putative anti-atherosclerotic effect.     

Repeated cadmium nebulizations induce pulmonary MMP-2 and MMP-9 production and emphysema in rats

This study describes induction of pulmonary inflammation, production of matrix metalloprotease of type 2 (MMP-2) and type 9 (MMP-9), and emphysema in cadmium (Cd)-exposed rats. Sprague-Dawley rats were randomly distributed into two groups: one placebo-exposed group undergoing saline (NaCl 0.9%) inhalation (n=30) and one Cd-exposed group undergoing cadmium (CdCl(2) 0.1%) inhalation (n=30). The animals of the placebo- and Cd-exposed groups were divided in five subgroups (n=6). Subgroups underwent either a single exposure of 1h or repeated exposures three times weekly for 1h during 3 weeks (3W), 5 weeks (5W), 5 weeks followed by 2 weeks without exposure (5W+2) or 5 weeks followed by 4 weeks without exposure (5W+4). Each animal underwent determination of enhanced pause (Penh) as index of airflow limitation prior to the first exposure as well as before sacrifice. The animals were sacrificed the day after their last exposure. The left lung was fixed for histomorphometric analysis (determination of median interwall distance (MIWD)), whilst bronchoalveolar lavage fluid (BALF) was collected from the right lung. BALF was analyzed cytologically, and MMP-2 and MMP-9 levels were determined by gelatine zymography. Twelve rats previously instilled with pancreatic elastase were used as positive emphysema controls and underwent the same investigations. Cd-exposure induced a significant increase of BALF macrophages, neutrophils and MMP-9 up to 5W+4, whereas MMP-2 gelatinolytic activity returned to baseline levels within 5W. MIWD was significantly increased in all repeatedly Cd-exposed groups and elastase-treated rats. Penh was increased in Cd-exposed rats after a single exposure and after 3W. MMP gelatinolytic activity was significantly correlated with macrophages, neutrophils and Penh. In repeatedly exposed rats, MIWD was positively and significantly correlated with MMP gelatinolytic activity, suggesting that increased MMP-2 and MMP-9 production favours the development of emphysema.     

Effects of dietary vitamin E,C and soybean oil supplementation on antioxidant enzyme activities in liver and muscles of rats

The effect of elevated levels of dietary vitamin E, C and a combination of vitamin E and C (E & C) with soybean oil on activities of antioxidant (AOE) enzymes important in the protection against lipid peroxidation was studied in male rats fed with vitamin C (12 mg/g), vitamin E (3.68 mg/g) or E & C (3.68 mg/kg + 12 mg/g) supplemented diets for 28 days. Catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activity in liver, pectoralis major (PM) and sartorius (S) muscles was increased significantly in rats fed with dietary vitamin C, E separately, and vitamin C & E combination, except, superoxide dismutase (SOD), which showed no alterations. These results clearly indicated that vitamin E & C separately and E & C together increased AOE activity in liver, PM and S muscle of rats. However, vitamin E and C combination enhanced AOE activity more significantly and our findings suggest the possible role of vitamin C & E and their combination in reducing the risk of chronic diseases related to oxidative stress.     

Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells

It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (MSP). Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24 h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas (p = 0.006, 0.007, 0.003, <0.001 and 0.001 respectively), as detected by immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.     

Cathepsin D and MMP-9 activity increase following a high intensity exercise in hind limb muscles of young rats

The influence of an intensive exercise regime on cathepsin D and MMP-9 activity in hind limb muscles was investigated. We hypothesized that high-intensity exercise would increase the number of these proteins, indicating their involvement in the pathogenesis of exercise-induced muscle injury. Muscle fibers from the gastrocnemius and soleus were used from young (6-mo-old) female rats (n = 6) who completed 10 consecutive days of treadmill running at high intensity (34 m min(-1) gradually up to 40 min per day), compared with nonrunning, age and sex-matched rats (n = 6). After a high-intensity exercise regime, cathepsin D activity significantly increased in the gastrocnemius (from 6.6 x 10(-3) to 10.7 x 10(-3) or 61% nM tyrosine x mg-1 protein x min-1) and the soleus (from 5.9 x 10(-3) to 8.9 x 10(-3) or 66%). The activity level of mRNA MMP-9, expressed as ng mg(-1) protein, increased in both muscles subjected to intensity running. The results of this study suggest that high-intensity running results in an elevation in the activity of lysosomal enzymes involved in matrix protein degradation.     

Acute and subacute toxicity and genotoxicity of schizonepetin,a naturally occurring monoterpene with antiviral activity

In the present study, the acute, subacute and genetic toxicity of schizonepetin was assessed. The median lethal dose (LD₅₀) of schizonepetin after oral administration was 478 mg/kg body weight in mice. Studies on dose toxicity were repeatedly conducted at 0, 60, 120, and 240 mg/kg bw/day in rats for 35 days after oral administration. Based on the results of this study, a dose level of 120 mg/kg bw/day is considered the no-observed-adverse-effect-level (NOAEL) in rats. Schizonepetin was negative in Salmonella typhimurium tester strains TA97, TA98, TA100, TA102 and TA1535, nonclastogenic in Chinese hamster lung (CHL) cells in the mammalian chromosome aberration test, and micronucleus formation were observed and no clinical signs or adverse effects were detected, and our results illustrated that schizonepetin is not genotoxic.     

Safety evaluation of steroidal saponin DT-13 isolated from the tuber of Liriope muscari (Decne.) Baily

Steroidal saponin DT-13 (25 (R, S)-ruscogenin-1-O-[β-d-glucopyranosyl - (1 → 2)] [β-d-xylopyranosyl-(1 → 3)]-β-d-fucopyranoside) is the main active component of the tube of Liriope muscari (Decne.) Baily and has been studied as a candidate drug for cancer metastasis. The objective of this study was to evaluate the safety of DT-13 systematically by genotoxicity and acute oral toxicity and subchronic 90-day oral gavage toxicity. Results of Ames test confirmed that DT-13 did not induce mutations in histidine auxotrophs Salmonella typhimurium (TA 97, TA 98, TA 100 and TA 102) both in the presence and absence of metabolic activation system at the doses of 0.05-500 μg/plate. Meanwhile, DT-13 did not induce clastogenicity at doses of 1250, 2500 and 5000 mg/kg in mouse micronucleus test. And the single oral dose of DT-13 at 5000 mg/kg did not produce mortality or significant changes in the general behavior and gross appearance of the internal organs of mice. In subchronic toxicity study, DT-13 was administrated to Sprague-Dawley rats via oral gavage at doses of 10, 60 and 360 mg/kg for 90 days. Necropsy, hematological and biochemical analysis, and histopathological examination did not reveal any remarkable and treatment related changes. In conclusion, DT-13 is of low toxicity at the tested doses.     

Effect of the herbicide pendimethalin on rat uterine weight and gene expression and in silico receptor binding analysis

The endocrine disrupting potential of the herbicide pendimethalin was investigated in vivo on the uterotrophic response and on the expression of estrogen-regulated genes examined by quantitative real-time RT PCR. Receptor binding characteristics of pendimethalin were analyzed by an in silico method. Pendimethalin (150, 225, 300 and 600 mg/kg/day) was administered by oral gavage to immature female rats for 3 days, with ethinylestradiol (0.001 mg/kg/day) as positive control. Pendimethalin caused a small but significant increase in absolute uterine weight at and above 300 mg/kg/day and in relative uterine weight at 600 mg/kg/day. Estrogen receptor (ER)-alpha mRNA levels were not affected, whereas ER-beta mRNA was up-regulated at the highest dose. Progesterone receptor mRNA level was not significantly changed, while insulin-like growth factor-I mRNA was reduced, significantly at 225 mg/kg/day to 65% of control. Androgen receptor (AR) mRNA showed a marked down-regulation at doses of 225 mg/kg/day and above. The expression pattern differed from that of ethinylestradiol. In silico analysis revealed potential binding of pendimethalin to ER-beta and AR, but virtually no binding to ER-alpha. These data demonstrate that pendimethalin exhibits estrogenic activity also in vivo. However, its uterotrophic effect, which is an ER-alpha-mediated response, is very small, and it appears that in vivo actions should rather be sought in ER-beta-regulated functions.     

Low doses of amphetamine lead to immediate and lasting locomotor sensitization in adolescent, not adult, male rats

Although there is much evidence for age differences in behavioural responses to psychostimulants in rats, the differential, lasting impact of drug exposures has rarely been investigated using direct comparisons of adolescent and adult rats. Male rats were pre-treated with 0.5 mg/kg amphetamine or saline on either postnatal days (P) 31 and P33 or P76 and P78, and locomotor activity was measured for 1 h. Adolescent, and not adult, rats showed a significant increase in distance traveled from the first to second pre-treatment. There was no evidence of sensitization of locomotor activity in either adolescents or adults on Challenge 1 to the same dose of amphetamine when tested 12 days later on P45 (late adolescence) or on P90. Rats that were pre-treated as adolescents exhibited locomotor sensitization to 1.5 mg/kg amphetamine as adults (P60) on Challenge 2, 27 days after pre-treatment, particularly in the group that had also received amphetamine on Challenge 1 at P45. Rats that were pre-treated as adults did not show sensitization on Challenge 2. The results suggest that the rapid adaptations to drug exposures in adolescence have greater consequences than identical treatment in adulthood, and highlight the unique vulnerability of adolescents to brief, low dose drug exposure.