Joseph F. Crivello

The effects of exercise training of different intensities on neuromuscular junction morphology

SummaryLittle is known about the effects of exercise training on neuromuscular junction morphology in skeletal muscle. The objectives of this investigation were: 1) to determine if exercise training would elicit changes in neuromuscular junction morphology, 2) to determine if exercise training of different intensities would evoke specific changes in neuromuscular junction morphology, and 3) to determine whether changes in neuromuscular junction structure occur independently of changes in muscle fibre type and size. Twenty-four age and size matched male Sprague-Dawley rats were randomly assigned to three groups: high-intensity trained (HIT), low-intensity trained (LIT), or untrained. Neuromuscular junction morphology of the soleus muscle was determined via immunofluorescent staining. Presynaptic acetylcholine vesicles were visualized with SV-2 antibody in conjunction with fluorescein isothiocyanate labelled secondary antibody. Postsynaptic acetylcholine receptors were identified with rhodamine labelled α-bungarotoxin. Laser scanning microscopy was used to produce images of synapses, which were used to quantitate the following: total area of SV-2 and α-bungarotoxin staining, density of acetylcholine vesicles and receptors, structural complexity, and synaptic coupling. To visualize nerve terminal branching, a smaller number of neuromuscular junctions were stained with C-2 antibody, which reacts with a neurofilament epitope, in conjunction with fluorescein isothiocyanate labelled secondary antibody. Total length of branching, number of branches, average length of branches, and ratio of secondary to primary branches per neuromuscular junction were determined. Citrate synthase activity, fibre type composition and fibre cross-sectional areas of the soleus muscle were assessed to determine the presence of a training effect in that muscle. Results indicate that training did induce hypertrophy of the neuromuscular junction that was independent of muscle hypertorphy. Although the HIT and LIT groups exhibited similar hypertrophic responses of the neuromuscular junction, the HIT group displayed more dispersed synapses than the LIT group. Neither exercise training program, however, resulted in altered densities of acetylcholine vesicles or receptors, nor did training significantly change synaptic coupling. Nerve terminal branching was also affected by exercise training. Neuromuscular junctions from the HIT group demonstrated a greater total length of branching, average length per branch, and number of finer, or secondary, branches than those of the LIT group.

Endurance and resistance exercise induce muscle fiber type specific responses in androgen binding capacity

This study examined the effects of different exercise training programs on androgen receptor content and receptor affinity to dihydrotestosterone in fast glycolytic (FG) and slow oxidative (SO) skeletal muscle fibers in rats. Twenty-four male Sprague-Dawley rats were equally divided into three groups: control, endurance exercise trained and resistance exercise trained. After the exercise programs were completed, the extensor digitorum longus (EDL), predominantly a FG muscle, and the soleus, predominantly a SO muscle, were isolated, weighted and both androgen receptor content and affinity to dihydrotestosterone were determined. Resistance training evoked a significant (P < 0.05) hypertrophic response in the soleus but not the EDL. Endurance training was not associated with any significant hypertrophy in either the soleus or the EDL. Neither the endurance nor the resistance training program resulted in changes in androgen receptor affinity to dihydrotestosterone. However, alterations in androgen receptor content were noted. The endurance training program resulted in a significant increase in androgen receptor content in the soleus, but no significant difference in the EDL. The resistance training program elicited a significant decrease in androgen receptor content in the soleus, and a significant increase in the EDL. These results indicate that different exercise stimuli induce changes in androgen receptor content that are specific to skeletal muscle fiber type.

Exercise-Induced Hormonal Changes and their Effects upon Skeletal Muscle Tissue

ConclusionThe neuroendocrine system plays an integral function in the development and maintenance of muscle tissue. Numerous investigations have confirmed the effects of both aerobic exercise and heavy resistance exercise upon the neuroendocrine system. Although there has been great progress in the area of exercise and neuroendocrinology, many questions regarding hormonal responses to exercise remain unanswered. In particular, there is a need for additional research that will reveal the specific mechanisms by which different exercise protocols induce hormonal responses.

Killifish metallothionein messenger RNA expression following temperature perturbation and cadmium exposure.

Abstract Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26°C or 10°C) and/or 6 ppb of waterborne cadmium chloride (CdCl2). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase–polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26°C compared with those exposed to 19°C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl2 compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone.

Elucidating the mechanism of action of tributyltin (TBT) in zebrafish

Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ(1) and zfERβ(2)) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ(1) and zfERβ(2) and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ(1) mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish.

Variation Among Four Health Indices in Natural Populations of the Estuarine Fish, Fundulus heteroclitus (Pisces, Cyprinodontidae), from Five Geographically Proximate Estuaries

Variation among four commonly used health indices was examined in the mummichog, Fundulus heteroclitus. The four indices were liver glycogen content (LGC), liver-somatic index (LSI), condition index (K) and RNA–DNA ratio. Fish were collected from five coastal locations in southeastern Connecticut. Fish health, as determined by these four indices, varied considerably among estuaries and between sexes. The relationship between each index and specimen length was significantly different among estuaries for either sex. When regressed against length, the slopes for the indices ranged from positive to negative. For each index, significant differences existed among some of the length-centered means at each estuary for either sex. Estuary rank for one index did not necessarily correlate with the estuary rank for another index. The significance of this variability and its impact on the use of the indices as bioindicators of environmental perturbation is discussed.

Effects of starvation on liver microsomal P450 activity in juvenile Pleuronectes americanus

Recent work has produced evidence to support the existence of a cytochrome P450 CYP2E1-like isoform in the marine fish, Pleuronectes americanus (winter flounder) (Wall K, Crivello JF. Toxicol Appl Pharmacol 1998;151:98-104). Starvation has been previously demonstrated to induce CYP2E1 activity (assayed as chlorzozazone-6-hydroxylase activity) in mammals and this study was undertaken to determine the effects of starvation on liver chlorzozaxone-6-hydroxylase and ethoxy-resorufin-O-deethylase activity (a CYP1A1 activity) in juvenile winter flounder liver microsomes. A 2-week starvation period resulted in a statistically significant increase in liver microsomal protein, and a decrease in liver lipid and liver glycogen. Ethoxy-resorufin-O-deethylase activity (pmol/min/mg microsomal protein) was reduced with starvation, chlorzoxazone 6-hydroxylase activity (pmol/min per mg microsomal protein) initially decreased but then increased over controls. When these activities were expressed per gm/liver (to account for the starvation-induced changes in liver microsomal protein), chlorzoxazone 6-hydroxylase activity doubled over control during starvation but ethoxy-resorufin-O-deethylase was not significantly changed. The effects of starvation on liver microsomal chlorzoxazone 6-hydroxylase and ethoxy-resorufin-O-deethylase activities are discussed in the context of the impact of physiological states on the ability of fish to detoxify marine xenobiotics.

Oxidative stress limits vitamin D metabolism by bovine proximal tubule cellsin vitro

When bovine proximal tubule cells are placed in primary culture, they are subject to elevated oxidative stress which acts to limit the expression of mitochondrial vitamin D3 1 alpha- and 24-hydroxylase activities. This increased oxidative stress was demonstrated by increased production of cell and mitochondrial membrane lipid hyperperoxides (LOOH). This increased production was prevented by the addition of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Cell and mitochondrial membrane LOOH increased from 1 to 2 pmol/mg protein on the day of plating to 70-90 pmol/mg protein after 6 days in culture. Pretreatment of cultures with BHA and BHT resulted in membrane LOOH of 15-20 pmol/mg protein after 6 days. Mitochondrial LOOH production was greater than total cell LOOH after 6 days. The increase in cellular oxidative stress was paralleled by decreases in both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. Mitochondrial hydroxylase activities were inversely proportional to the increase in mitochondrial membrane LOOH production. Mitochondrial cytochrome P-450 content, determined spectrophotometrically, was decreased over time in culture. Mitochondrial cytochrome P-450 content determined by a specific polyclonal antibody in an enzyme-linked immunosorbant assay also decreased over time in culture. Specificity of polyclonal antibodies, raised against rat liver microsomal cytochrome P-450 RLM5, was demonstrated by the immunosequestration of both 1 alpha- and 24-hydroxylase activities from a partially purified preparation of renal mitochondrial cytochrome P-450. BHA showed the loss of 1 alpha- and 24-hydroxylase activities and mitochondrial P-450 content measured by all criteria. These experiments indicate that oxidative stress-mediated changes in hydroxylase activities are mediated directly by changes in hydroxylase content and not at distal sites. A partially purified preparation of bovine proximal tubule mitochondrial cytochrome P-450, with purified renal ferredoxin, ferredoxin reductase, and NADPH, expressed both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. LOOH, derived from mitochondrial membranes of 5-day-old cultures, when added to this mixture, caused a dose-dependent decrease in both activities. These experiments suggested that an increase in mitochondrial LOOH production resulted in a loss of 1 alpha- and 24-hydroxylase activities. 1 alpha-Hydroxylase was more sensitive to the effects of LOOH treatment than 24-hydroxylase. At a ratio of LOOH:P-450 of 5:1 (molar), all 1 alpha-hydroxylase activity was lost but 50% of the 24-hydroxylase activity remained.(ABSTRACT TRUNCATED AT 400 WORDS)

MULTIPLE PATHWAYS OF PROSTATE CARCINOGENESIS ANALYZED BY USING CULTURED CELLS ISOLATED FROM RATS TREATED WITH N-METHYL-N-NITROSOUREA AND TESTOSTERONE

Treatment of rats with N‐methyl‐N‐nitrosourea (MNU) and testosterone results in a high incidence of metastasizing dorsolateral prostate tumors. In previous studies, a high frequency (≥70%) of a G35 → A transition mutation at the second position of codon 12 of the Ki‐ras oncogene was found in these tumors. This was confirmed in the study reported here, and the frequency of this mutation appeared similar in tumors induced in four different rat strains, regardless of differences in sensitivity among these strains to the induction of prostate cancers by MNU and testosterone: Wistar Furth (62% incidence of grossly visible prostate tumors) > Lobund Wistar (55%) > Fisher 344 (40%) > Copenhagen (37%). A method was developed to isolate and separately culture epithelial and stromal cells from these rat prostate carcinomas. Of 20 primary cell cultures established from histologically confirmed rat prostate carcinomas, 19 (95%) displayed one or more of the following characteristics: the Ki‐ras mutation (17 of 20; 85%), anchorage‐independent growth in soft agar at early passage (12 of 20; 60%), or tumorigenicity at later passage (eight of eight; 100%). One epithelial cell culture and all five stromal cell cultures established from prostate tumors had none of these characteristics. Epithelial cultures that had the Ki‐ras mutation and grew in soft agar constitute the predominant genotype/phenotype (55%), cultures with the mutation that did not grow in soft agar were less frequent (30%), 10% of the cultures had neither characteristic, and only one grew in soft agar but did not have the mutation. These findings suggest that there are at least two and perhaps more different molecular pathways of prostate carcinogenesis in rats treated with MNU plus testosterone. Furthermore, these data suggest that these pathways and the mechanisms determining strain differences in sensitivity to prostate cancer induction are unrelated. Mol. Carcinog. 25:179–186, 1999.

Variations in Physiological Biomarkers among Mummichogs Collected from Connecticut Salt Marshes

Abstract In fall 1998, a sample of 716 mummichogs Fundulus heteroclitus was collected from seven Connecticut salt marshes as part of an estuary biomonitoring program. The collection sites consisted of one relatively unpolluted reference site and six others with varying pollution loads. Several physiological biomarkers (body weight and length, liver weight, RNA:DNA ratio, liver somatic index, liver glycogen content, and condition index) were determined and compared with the known pollution characteristics at the collection sites. When normalized to the levels found at the reference site, average health indices were 12% lower at the low- to moderate-impact sites and 30% lower at the high-impact sites. Average health indices were more strongly depressed in female fish (18% and 35%) than in male fish (0% and 15%). For individual fish, body weight was strongly correlated with length, liver weight, condition index, and the RNA:DNA ratio. Liver glycogen content was not correlated with any of the other health ind...