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Biomedical subjects

M G Miller

Publications and source records attributed to M G Miller.

At least 19 recordsLinked to original sources
PubMed2002

Stage-specific effects of the fungicide carbendazim on Sertoli cell microtubules in rat testis.

The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ.

Administration, Oral↗
PubMed2001

The role of GTP Binding and microtubule-associated proteins in the inhibition of microtubule assembly by carbendazim.

The fungicide carbendazim (CBZ) is known to disrupt microtubular structures in the testis and to cause testicular toxicity in rats. To investigate the mechanism underlying the toxicity of CBZ, tubulin and microtubule-associated proteins (MAPs) were isolated from rat testis and brain using two techniques. The effects of CBZ on MT assembly were compared with the known microtubule (MT) disruptors, colchicine and nocodazole. CBZ (100 microM) had no effect on the assembly of MTs from MAP-containing tubulin isolated with one cycle of glycerol-dependent assembly and disassembly while colchicine (40 microM) and nocodazole (12.5 microM) strongly inhibited the assembly reaction. Similarly, formation of MTs from tubulin prepared with two cycles of glycerol-dependent assembly was strongly inhibited by colchicine and nocodazole but only weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP-free tubulin isolated with glutamate. However, the inhibition by CBZ was reversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled MTs caused immediate depolymerization, whereas CBZ did not directly cause depolymerization. However CBZ was an effective inhibitor of the polymerization of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data suggest that CBZ interferes with initial events of MT polymerization, specifically GTP binding, and that MAPs moderate this effect.

Animals↗
PubMed2001

alpha-Chlorohydrin inhibits glyceraldehyde-3-phosphate dehydrogenase in multiple organs as well as in sperm.

Numerous studies have documented inhibitory effects of alpha-chlorohydrin (ACH) on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in spermatozoa. A sperm-specific G3PDH isoform has been described. The possibility that ACH may inhibit G3PDH in cell types other than sperm was investigated in this work. In addition, the onset of ACH-induced epididymal toxicity was described. Changes to epididymal histology occurred 6 h following a single dose of ACH (50 mg/kg po) and were confined to the proximal initial segment. By 24 h, no epithelial cells lined the basement membrane of that region. Three h after ACH administration (50 mg/kg po), G3PDH activity was significantly decreased in sperm (85%) as well as in kidney (31%), liver (49%), and epididymis (35%). Enzyme activity remained inhibited at 6 and 24 h. G3PDH was immunolocalized in the epididymis and staining was highest in the efferent ducts and initial segment as well as in smooth muscle. Since G3PDH is a microtubule-associated protein and microtubule-dependent endocytosis occurs in the epididymis, beta-tubulin was also immunolocalized. beta-tubulin densely stained the apical region of initial segment and caput epithelial cells. Disruption of beta-tubulin immunostaining correlated with the localization and onset of the lesion. Co-localization of G3PDH and beta-tubulin immunostaining was not observed although both antibodies most densely stained the initial segment. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition. Moreover, ACH inhibition of G3PDH is not confined to sperm, although the sperm enzyme is most sensitive to inhibition.

Animals↗
PubMed2001

Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin.

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine on microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymerized tubulin pools were separated and tubulin was quantified using a competitive ELISA. Carbendazim and colchicine caused extensive microtubule depolymerization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained relatively constant after carbendazim treatment but decreased twofold after colchicine treatment. To determine if modifications to tubulin may be associated with polymerization status, tubulin pools were analyzed by immunoblotting. Acetylated alpha-tubulin and betaIII-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha-tubulin (52 kDa) was localized in both tubulin pools and had decreased tyrosination in the microtubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated alpha-tubulin and general alpha-tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alpha-tubulin isoform) lost tyrosination, yet was still present in the microtubule pool based on detection with the general alpha-tubulin antibody, after carbendazim treatment. Similar effects were seen with colchicine, although loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymerization of microtubules.

Acetylation↗
PubMed2000

Health-related physical fitness knowledge of student allied health professions.

Each allied-health profession has their own particular expertise but also shares some commonalities. One such commonality should be knowledge of health-related physical fitness relating to the health and well-being of individuals. Although the benefits of health-related physical fitness have been well documented, few studies have examined the level of health-related physical fitness knowledge among allied-health professions. Therefore, the purpose of this investigation was to assess the health-related physical fitness knowledge of three allied health professions using a 40 item multiple-choice test designed to assess knowledge in five domains of health-related physical fitness. Results indicated that student athletic trainers scored significantly higher on the post-test versus pre-test. On the post-test, athletic training and physical therapy groups scored significantly higher than the nursing group. The information from this study may be valuable in aiding educators in developing appropriate curricula to better prepare students for their role as allied health professionals.

Allied Health Occupations↗
PubMed1999

Continuous subcutaneous infusion of morphine vs. hydromorphone: a controlled trial.

Seventy-four patients were included in a double-blind, randomized, controlled trial comparing the analgesic efficacy and adverse effects of hydromorphone and morphine delivered by continuous subcutaneous infusion. Patients completed the Memorial Pain Assessment Card and a checklist of opioid-related adverse effects immediately before commencing subcutaneous infusion and 24, 48, and 72 hours later. An assessment tool was developed for the 60 patients who were too ill to complete their own questionnaire. The tool demonstrated excellent inter-rater reliability. Thirty-four percent of patients in the hydromorphone group and 27% of those in the morphine group died before completion of the study (P = 0.66). The hydromorphone group required more analgesia for breakthrough pain in the first 24 hours of the study (P = 0.03) and had a greater improvement in the behavior of frowning on movement and the comfort visual analogue scale (P = 0.08) over the course of the study. Adverse effects were rare and similar in both groups. This study found hydromorphone to be at least as effective as morphine when delivered by continuous subcutaneous infusion.

Adult↗
PubMed1999

Rat testis motor proteins associated with spermatid translocation (dynein) and spermatid flagella (kinesin-II).

In this study, we report sites in the seminiferous epithelium of the rat testis that are immunoreactive with antibodies to the intermediate chain of cytoplasmic dynein and kinesin II. The study was done to determine whether or not microtubule-dependent motor proteins are present in Sertoli cell regions involved with spermatid translocation. Sections and epithelial fragments of perfusion-fixed rat testis were probed with an antibody (clone 74.1) to the intermediate chain of cytoplasmic dynein (IC74) and to kinesin-II. Labeling with the antibody to cytoplasmic dynein was dramatically evident in Sertoli cell regions surrounding apical crypts containing attached spermatids and known to contain unique intercellular attachment plaques. The antibody to kinesin II reacted only with spermatid tails. The levels of cytoplasmic dynein visible on immunoblots of supernatants collected from spermatid/junction complexes treated with an actin-severing enzyme (gelsolin) were greater than those of controls, indicating that at least some of the dynein may have been associated with Sertoli cell junction plaques attached to spermatids. Results are consistent with the conclusion that an isoform of cytoplasmic dynein may be responsible for the apical translocation of elongate spermatids that occurs before sperm release. Also, this is the first report of kinesin-II in mammalian spermatid tails.

Animals↗
PubMed1999

Teaching medical students cancer risk reduction nutrition counseling using a multimedia program.

BACKGROUND AND OBJECTIVES: There are many barriers to medical students receiving education about the linkage between nutrition and cancer, including the lack of role models and teachers and insufficient curricular time. We tested the use of a multimedia program as a possible solution to teaching diet-risk assessment and counseling skills. METHODS: Images of Cancer Prevention, The Nutrition Link is a CD-ROM multimedia program that was developed and evaluated by 147 medical students. Pre-use and post-use surveys, computer log files, and recorded response sessions were used to determine the learner's 1) ease in using the program, 2) attitudes about the treatment of the content, 3) knowledge gain, and 4) attitudes about the role of physicians in nutrition assessment and counseling for cancer risk reduction. RESULTS: Students improved their knowledge of dietary guidelines for cancer risk reduction and made positive changes in their attitudes toward the role of physicians in dietary counseling. However, most students reported that they would not use the program unless it was required that they do so. CONCLUSIONS: The multimedia program was successful; it affected students' knowledge and attitudes concerning nutrition as a modifiable risk factor for some cancers. In addition, the design and delivery of the multimedia product was positively reviewed by the students for ease of access, message design, individualized instruction, and flexibility. Despite these favorable ratings, it was not clear that students would use the program unless required to do so.

Health Knowledge, Attitudes, Practice↗
PubMed1999

Comparison of human and rat metabolism of molinate in liver microsomes and slices.

Molinate undergoes oxidative metabolism forming either ring-hydroxylated metabolites or molinate sulfoxide. Our previous studies strongly implicated the sulfoxidation pathway in molinate-induced testicular toxicity. The present study compares the metabolic capability of rat and human liver microsomes and slices to form either nontoxic ring-hydroxylated metabolites of molinate or the toxic metabolites derived from the sulfoxidation of molinate. Km and Vmax values indicate that sulfoxidation would be the preferred high-dose pathway whereas hydroxylation would predominate at low dose levels in both species. Examination of phase II metabolism of molinate in liver slices reveals greater detoxification of molinate sulfoxide by glutathione conjugation in humans with rats forming less conjugate. Oxidative metabolism of molinate in both rats and humans appears to be mediated by cytochrome P-450 and not flavin monooxygenases as indicated by the use of metabolic inhibitors. Overall, the metabolism of molinate would be via the nontoxic hydroxylation pathway in both species at low doses whereas at high doses, where sulfoxidation would predominate, the human is more capable than the rat to detoxify via glutathione conjugation.

Animals↗
PubMed1998

Testicular toxicity of molinate in the rat: metabolic activation via sulfoxidation.

Molinate is a thiocarbamate herbicide widely used in rice culture. Studies conducted for regulatory purposes have indicated that molinate exposure causes male reproductive damage in rats. The present study describes the testicular lesion after administration of single doses of molinate. The hypothesis that a metabolite of molinate is responsible for testicular toxicity was also investigated. Testicular damage was evaluated histopathologically in Sprague-Dawley rats 48 h and 1, 2, and 3 weeks after administration of molinate (100-400 mg/kg i.p.). No testicular damage was seen at any time point at the 100 mg/kg dose level. Damage was first seen 1 week after 200 mg/kg and 48 h after 400 mg/kg. The lesion was characterized by Sertoli cell vacuolation, failed spermiation, and phagocytosis of spermatids particularly evident at Stages X and XI. With increasing time, damage progressed until disorganization of the seminiferous epithelium was extensive, multinucleated giant cells were numerous, and neither spermatozoa nor late step spermatids were present. At 3 weeks after administration of the two higher-dose levels, germ cells in the seminiferous tubules were almost completely absent. Administration of the sulfoxide metabolite of molinate (200 mg/kg i.p.) caused testicular damage similar in severity to that seen at the 400 mg/kg dose level for the parent compound, indicating that it was more potent as a testicular toxicant. In vitro metabolism studies using liver and testis microsomes found that the major metabolite in both preparations was molinate sulfoxide. Testis microsomes produced only slightly less sulfoxide when compared with liver microsomes. Molinate was also metabolized via ring hydroxylation to form small amounts of hydroxymolinate. The amount of hydroxymolinate was substantially less in testis microsomes. Overall, these data indicate that sulfoxidation of molinate plays a role in molinat-induced testicular toxicity. Moreover, molinate is metabolized readily by both liver and testis microsomal enzymes, suggesting that the molinate toxic metabolite could be formed in the testis in close proximity to its site of action.

Animals↗
PubMed1998

Identification of a carboxylesterase as the major protein bound by molinate.

Molinate, a herbicide widely used on rice, has been previously shown to cause testicular toxicity when a single dose is administered to Sprague-Dawley rats. The sulfoxide metabolite of molinate also was capable of eliciting testicular damage but at lower dose levels than molinate, suggesting that metabolic activation via sulfoxidation could be important in testicular toxicity. Both the sulfoxide and sulfone metabolites of molinate are electrophilic and molinate covalent binding to cellular macromolecules has been attributed to formation of these reactive metabolites. The present study has investigated the nature of the binding reaction of 14C-molinate as well as 14C-molinate sulfoxide and 14C-molinate sulfone in liver and testis microsomal preparations. All three compounds in preparations from both tissues bound extensively and tightly to only one protein of approximately 60 kDa molecular weight on SDS-PAGE. Isoelectric focusing PAGE revealed a pI of approximately 6.0 and native PAGE analysis revealed a native molecular weight of 180 kDa. These data, along with the ability of phenylmethylsulfonyl fluoride to block binding of the 14C-molinate, suggested the molinate-bound protein was an esterase. The protein was purified to homogeneity and MALDI-TOF mass spectral analysis was consistent with Hydrolase A, a carboxylesterase present in both liver and testis. N-terminal sequence analysis revealed 100% homology with Hydrolase A for the first 17 residues. The effect of molinate administration on in vivo esterase activity was assessed both by enzymatic measurement and by histochemical measurement. Molinate treatment caused a marked inhibition of nonspecific esterase activity in both liver and testis. In the testis, histochemical staining showed the esterase activity inhibited by molinate was localized primarily to the Leydig cell, consistent with the localization of Hydrolase A. From these data, it is proposed that molinate-induced inhibition of esterase activity in the Leydig cell could inhibit the mobilization of cholesterol esters required for testosterone biosynthesis.

Amino Acid Sequence↗
PubMed1997

1,3-Dinitrobenzene metabolism and toxicity in seminiferous tubules isolated from rats of different ages.

Previous in vivo studies in rats have shown that susceptibility to 1,3-dinitrobenzene (DNB)-induced testicular damage increases with age. The present study has used an in vitro approach to investigate the possibility that differences in testicular metabolism contribute to the age-related differences in toxicity. Seminiferous tubules were isolated from Sprague-Dawley rats (30, 75 and 120 days old) and incubated with 100 microM DNB for 22 h. Formation of metabolites and tubular levels of ATP and glutathione (GSH) were monitored over time. There was no difference in seminiferous tubule metabolic capacity among the three ages of rats examined. After 22 h of incubation with DNB, ATP levels were 20-30% of control and GSH levels were 70-90% of control, but neither parameter showed an age-related difference in decline. Based on these biochemical indicators of cell health, this study would suggest that the lack of testicular toxicity in young animals in vivo may be due to the previously described shorter half-life with consequent reduced exposure of the testis to DNB and that the age-related increase in severity of lesion between 75 and 120 days of age cannot be explained by differences in tubular metabolism of DNB or whole-animal toxicokinetics.

Adenosine Triphosphate↗
PubMed1997

Role of testis exposure levels in the insensitivity of prepubertal rats to carbendazim-induced testicular toxicity.

Our recent studies have indicated that benomyl (BNL)-induced testicular toxicity is mediated by its major metabolite carbendazim (CBZ). The present study has used CBZ to investigate hypotheses that could explain prepubertal insensitivity to BNL. When CBZ (164 mg/kg intraperitoneally) was administered to postpubertal and prepubertal rats, it caused little testicular damage in prepubertal rats, but in adult rats, sloughing of the seminiferous epithelium resulted. When the inhibitory effect of CBZ on prepubertal testicular microtubule assembly was compared with that on postpubertal assembly, the IC50 values were very similar. Pharmacokinetic studies revealed that blood levels of CBZ were comparable in the two age groups; however, higher levels of CBZ were found in the adult testes (210.52 nmol/g wet wt) in comparison with young testes (67.77 nmol/g wet wt). These data suggest that delivery to and/or retention of CBZ in the testis may play a role in the age-dependent differences in susceptibility to CBZ toxicity. When CBZ was administered intratesticularly to reach levels sufficient to cause damage, the young animals did show an increased incidence of vacuolization and detachment of the seminiferous epithelium; however, in contrast to the older animals, sloughing of the seminiferous epithelium was not observed in the prepubertal animals. Overall, the low levels of CBZ measured in the testes of prepubertal animals offer a partial explanation for the insensitivity of young animals to CBZ-induced testicular toxicity following intraperitoneal administration. A differential responsiveness between the two age groups is also likely, however, since prepubertal animals lack elongated spermatids and it is sloughing of this cell type that characterizes CBZ-induced testicular toxicity in the adult.

Animals↗
PubMed1997

The role of the benomyl metabolite carbendazim in benomyl-induced testicular toxicity.

The present study has investigated the role of benomyl (BNL) vs carbendazim (CBZ) in BNL-induced testicular toxicity. Equivalent molar concentrations of BNL and CBZ were administered to rats intraperitoneally (859 mumol/kg) or by direct injection into the testis (1.37 mumol/testis). Whereas no significant testicular damage was observed both 1 and 2 hr after BNL administration by the ip route, CBZ administration resulted in sloughing of the seminiferous epithelium after 1 hr, which increased in severity at the 2-hr time point. Intratesticular treatment of BNL caused little testicular damage after 1 hr whereas an equimolar amount of CBZ elicited severe disruption of the seminiferous epithelium. Testicular levels of CBZ and BNL were measured at various times after both routes of administration. The AUC from the concentration of CBZ in the testis vs time plot showed an excellent relationship to the number of tubules which exhibited slouging. The BNL AUC also showed a straight-line relationship to severity of lesion. However, when the contribution of CBZ to the BNL response was subtracted, no effect of BNL was discernible. The effect of BNL and CBZ on testicular microtubule assembly was then investigated. IC50 for CBZ was 5 microM and that for BNL was 75 microM. Again, the effect of BNL on microtubule assembly could be largely accounted for by the presence of the CBZ breakdown product. These results strongly suggest that the BNL metabolite CBZ, and not BNL itself, is the mediator of BNL-induced testicular toxicity and inhibitor of testicular microtubule assembly.

Animals↗
PubMed1996

Trout liver slices for metabolism and toxicity studies.

Aquatic species are increasingly used in metabolism and toxicity studies, both from the perspective of potential for chemical exposure and usefulness as nonmammalian model systems. In the present study, trout liver slices were compared with freshly isolated trout hepatocytes with regard to metabolic capabilities and biochemical indices of cell health. Liver slices were also used to discern toxicant-induced changes in liver cell histology. Levels of ATP and glutathione were similar between liver slice and isolated hepatocyte preparations. The cytochrome P450-dependent rate of formation of biphenyl metabolites was 0.48 +/- 0.04 nmol/min/mg protein in slices and 0.43 +/- 0.06 nmol/min/mg protein in isolated cells. 7-Ethoxycoumarin metabolism was also comparable between preparations (1.36 vs. 1.22 nmol/min/mg protein). For conjugative metabolism, glucuronidation of 7-hydroxycoumarin or 1-naphthol did not differ in the two in vitro systems. However, neither slices nor isolated hepatocytes sulfated 7-hydroxycoumarin, whereas 1-naphthylsulfate represented as much as 20% of total 1-naphthol metabolites in both preparations. Histological evaluation of control liver slices after a 24-hr incubation indicated only minor changes. Response to the hepatotoxicants allyl formate and allyl alcohol was evaluated in slices only. Both compounds, after a 4-hr treatment and at concentrations between 0.1 and 1.0 mM, caused extensive depletion of glutathione, but ATP levels were unchanged. Histopathological damage was seen in slices incubated for 24 hr with either toxicant, but was most pronounced with allyl alcohol. These data indicate that liver slices are an excellent in vitro model for metabolism and toxicity studies in aquatic species.

1-Propanol↗