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Biomedical subjects

M G Miller

Publications and source records attributed to M G Miller.

87 records · Page 5Linked to original sources
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Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function.

Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.

Animals↗
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alpha-chlorohydrin induced changes in sperm fertilizing ability in the rat: association with diminished sperm ATP levels and motility.

In the present study, alpha-chlorohydrin (ACH) (5, 10, 25, 50 and 75 mg/kg, po) was administered to rats and the effects on sperm ATP levels, sperm motility, and the ability of sperm to bind and penetrate rat oocytes were determined. Groups of rats were killed 5 days and 3 h following treatment. At both time points, sperm from ACH-treated rats (>/=10 mg/kg) had significantly lower levels of ATP when diluted in media containing glucose. No diminution of ATP was seen in sperm diluted in phosphate-buffered saline (PBS). Computer analysis of sperm motility indicated that straight-line velocity (VSL) was the most sensitive parameter to ACH treatment and was significantly decreased in rat sperm three hours after ACH exposure (25 mg/kg). A clear drop in percent penetration (35% vs. 85% in control) of zona-free rat oocytes by rat sperm of both ACH groups was observed at 10 mg/kg. Higher dose levels produced no significant further decrease in percent penetration. Overall, the fertilizing ability of sperm was highly sensitive to ACH doses that caused minor but significant changes in sperm ATP levels and no significant changes in motility. These data are consistent with the spermatozoan's need for an uncompromised energy supply to maintain its ability to bind and penetrate the oocyte.

Adenosine Triphosphate↗
PubMedDate not supplied

Metabolism and testicular toxicity of 1,3-dinitrobenzene in the rat: evaluation of the stage-synchrony model.

Because many testicular toxicants cause damage to specific stages of spermatogenesis, the present study has investigated the utility of a model in which the testis is synchronized to contain only a few closely related spermatogenic stages. The susceptibility of different stages to 1,3-dinitrobenzene (1,3-DNB) toxicity was investigated in rats, the testes of which had been stage synchronized by a vitamin A depletion/repletion (VADR) procedure. 1,3-DNB (25 mg/kg, IP) or vehicle was injected 58, 61, or 78 d after vitamin A readministration, and testicular histopathology was evaluated 48 h later. At the time of sacrifice, testes in the three groups were synchronized to stages I-VI, VII-IX, or X-XIV+I. The data indicated that tubules in all stages of spermatogenesis, in both synchronized and unsynchronized animals, demonstrated histopathologic changes in response to 1,3-DNB. However, the lesion seen in synchronized animals was more severe and less stage specific than that seen in weight-matched, unsynchronized animals. This increase in degree of susceptibility could be partially explained by differences in toxicokinetics. Stage-synchronized testes could provide unique insights into stage-specific cellular and molecular events, especially for in vitro studies where the stage enrichment could be maximally exploited. However, results obtained from in vivo toxicity studies using animals subjected to VADR should be interpreted carefully in light of the confounding physiologic/metabolic perturbations potentially induced by the VADR procedure.

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Species difference in 1,3-dinitrobenzene testicular toxicity: in vitro correlation with glutathione status.

It has been previously shown that 1,3-dinitrobenzene (DNB) causes testicular damage to the rat but not the hamster. The present study of DNB's mechanism of toxic action has exploited this species difference in susceptibility. Seminiferous tubules were isolated from Golden Syrian hamsters and incubated with 100 microM DNB or vehicle for 22 h. (A similar study with rat tubules has been published.) Formation of DNB metabolites were monitored over time; hamster tubules had a greater capacity than rat tubules for reductively metabolizing (activating) DNB. However, hamster tubules did not show the marked DNB-induced ATP depletion seen in rat tubules. Levels of mitochondrial glutathione and activities of enzymes that protect against oxidative stress were measured in both rat and hamster tubules. The observed differences in the capacity for detoxification of oxidants may underlie the difference in susceptibility to DNB-induced testicular toxicity between these species.

Adenosine Triphosphate↗
PubMedDate not supplied

Germ cell apoptosis in rat testis after administration of 1,3-dinitrobenzene.

The present study investigated the occurrence of apoptosis in rat testis 6, 12, 24, or 48 h after administration of 1,3-dinitrobenzene (DNB)(25 mg/kg, i.p.). Apoptotic cells were visualized using TUNEL in situ detection. In untreated control animals, occasional tubules contained a few apoptotic nuclei. After DNB treatment, more tubules were apoptotic and more germ cells within individual tubules were affected. The increase in incidence and severity of apoptosis was statistically significant 24 and 48 h after exposure. Spermatocytes were the primary cell population affected. Apoptosis was primarily present in Stages VI-VIII and IX-XIII. DNA ladders, characteristic of apoptosis, were clearly present by 24 h after DNB administration, faint at 12 h, and not present in the control or at 6 h. Although initiating mechanisms may be very different for different chemicals, apoptosis is a common and late manifestation of the testicular response to numerous toxicants.

Animals↗
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In vitro fertilization after in vivo treatment of rats with three reproductive toxicants.

One objective of these experiments was to establish a sensitive assay to evaluate fertilizing potential of rat gametes in vitro. A second objective was to evaluate this in vitro fertilization (IVF) assay as a method to detect in vivo effects of reproductive toxicants on male and female gametes using three known reproductive toxicants as model systems. The IVF assay with zona-free oocytes was more precise than the assay with cumulus-intact oocytes in these studies (coefficients of variation of 8.7 and 14.4%, respectively). Sperm fertilizing potential for zona-free oocytes was reduced by treatment of rats with m-dinitrobenzene (10-10 000 microg/kg) and ethylene glycol monomethyl ether (50-100 mg/kg) that had no effect on sperm motility. Molinate (60 mg/kg for 5 days) reduced sperm fertilizing potential concurrently with reductions in sperm motility. Neither molinate (60 mg/kg for 5 days) nor dinitrobenzene (0.002% in the drinking water for 14 days) administered to females seemed to affect the fertilizability of their oocytes. Ethylene glycol monomethyl ether treatment (0.15-0.25% in the drinking water for 14 days) reduced the number of ovulated oocytes. IVF is a means to evaluate toxicant effects on female gametes and demonstrates sperm's ability to interact with the oocyte plasma membrane.

Animals↗
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Virtual seminars for medical nutrition education: case example.

Nutrition educators are exploring Web-based strategies for delivering course material. This report describes an effort to use computer-mediated communication between medical students and experts. A "virtual seminar" designed to help students apply nutrition knowledge to patient care was added to an existing traditional clinical skills course. There are few models for online nutrition education. Since these efforts are expensive in time, money, or both, it is important to share preliminary findings, including new methods of evaluating participation and effectiveness. Although medical student activity in the seminar was greater than anticipated, the return rate for the online evaluation survey was disappointing.

Adult↗
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Application of ELISA techniques to metabolic disposition studies for 1,3-dinitrobenzene: comparison with HPLC and radiochemical methods.

An enzyme-linked immunosorbent assay (ELISA) is reported for the detection and quantitation of 1,3-dinitrobenzene, a widely used chemical intermediate in industrial syntheses and potent testicular toxicant in rats. Radiolabeled [14C]-1,3-dinitrobenzene (25 mg/kg, ip) was administered to rats, and the blood disappearance curve generated with the ELISA assay was compared with HPLC separation and quantitation by using both UV and radiochemical methods. An excellent correlation was found between the two methods. Matrix effects from the blood samples were minimal. Cross reactivity with metabolites of 1,3-dinitrobenzene occurred only at levels that were 3 orders of magnitude greater than for the parent compound. The specificity of the ELISA for 1,3-dinitrobenzene was further demonstrated with a variety of other nitroaromatic compounds, of which only 2,4-dinitrotoluene showed significant cross reactivity. The sensitivity and specificity of the 1,3-dinitrobenzene ELISA demonstrate the usefulness of an approach which, if verified with conventional analytical methods, allows rapid and inexpensive measurement of xenobiotic levels in biological samples.

Animals↗
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Xenobiotic metabolizing enzyme activities in rat, mouse, monkey, and human testes.

The capacity of the testis to metabolize xenobiotics has been proposed to play a role in the susceptibility of different species to testicular toxicity. Since species differences in testicular xenobiotic metabolizing enzyme activities are not well documented, the primary objective of the present study was to compare enzyme activities in subcellular fractions prepared from rat, mouse, monkey, and human testes. In microsomal fractions, enzyme activities measured were pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-dealkylase (EROD), and epoxide hydrolase (mEH). In cytosolic preparations, epoxide hydrolase (cEH) and glutathione S-transferase (cGST) activities were measured. PROD activity was not detectable in any of the species studied, while it was readily detected in liver microsomes used as a positive control. Although EROD activity was low, it was measurable in testicular microsomes from rat and mouse, but not monkey or human. No marked species differences in cEH activity were found. In contrast, mEH activity was low in the monkey, intermediate in the rat, and highest in the human and mouse. cGST activity was significantly lower in the two primate species compared with the rat and the mouse. The levels of activity of the xenobiotic metabolizing enzymes studied were generally more than an order of magnitude lower in the testis as compared to the liver. However, in rat and mouse, the levels of mEH and cGST activities in testis were relatively similar to hepatic levels. Overall, these data indicate that species differences in capacity to metabolize xenobiotics may play a role in differential sensitivity to testicular toxicants.

Adult↗
PubMedDate not supplied

Metabolism and pharmacokinetics of 1,3-dinitrobenzene in the rat and the hamster.

Recent studies have documented that the Sprague-Dawley rat is markedly more sensitive than the Golden Syrian hamster to 1,3-dinitrobenzene (1,3-DNB)-induced testicular toxicity and methemoglobinemia. The present studies have investigated the possibility that differences in 1,3-DNB metabolism and pharmacokinetics could explain this species difference. [14C]1,3-DNB (25 mg/kg, ip) was administered to both species at a dose known to induce a testicular lesion in the rat and levels of 1,3-DNB and its metabolites were measured in blood and urine. Elimination of 1,3-DNB from the blood was initially rapid, followed by a second, much slower phase. Peak blood levels of 1,3-DNB were very different between the two species, with the hamster reaching levels only one-half those found in the rat (46.3 vs. 99.5 nmol/ml, respectively). Administration of a 50-mg/kg dose to the hamster resulted in 1,3-DNB blood levels which were similar to those found in the rat at 25 mg/kg. Since no obvious testicular toxicity is apparent in the hamster even at the higher dose level, a direct effect of 1,3-DNB on the testes seems unlikely, although it is possible that hamster cells inherently lack sensitivity to toxicity. Other major differences between the two species were a more rapid initial elimination rate and much higher blood levels of nitroaniline in the rat. Analysis of urinary metabolites revealed that the rat excreted more unconjugated metabolites and less phenolic metabolites compared with the hamster. Overall, the data indicate that the capacity to form reductive metabolites may play an important role in susceptibility to toxicity.

Animals↗
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Relationship between sulfotransferase activity and susceptibility to acetaminophen-induced liver necrosis in the hamster.

The effect of pretreatments which modulate acetaminophen sulfotransferase activity on the hepatotoxicity of acetaminophen have been examined in the hamster. Co-administration of sodium sulfate modestly enhanced the formation of acetaminophen sulfate, but provided little protection against liver injury. In isolated hepatocyte studies, sodium sulfate enhanced the Vmax of acetaminophen sulfotransferase activity, but did not alter the apparent Km toward acetaminophen. Administration of 2,6-dichloro-4-nitrophenol with acetaminophen selectively depressed acetaminophen sulfate formation in vivo and significantly exacerbated acetaminophen hepatotoxicity. In kinetic studies using isolated hamster hepatocytes, 2,6-dichloro-4-nitrophenol competitively inhibited acetaminophen sulfotransferase with a Ki of 2.5 X 10(-6) M. The data indicate that in the hamster, acetaminophen sulfotransferase activity plays a relatively minor role in the modulation of acetaminophen hepatotoxicity, and that, at hepatotoxic doses, the capacity limitation on this enzyme system is determined to a greater extent by its Km (app) value than by limitation in cofactor (3'-phosphoadenosine 5'-phosphosulfate) availability.

Acetaminophen↗
PubMedDate not supplied

Effect of L-ascorbic acid on acetaminophen-induced hepatotoxicity and covalent binding in hamsters. Evidence that in vitro covalent binding differs from that in vivo.

The known potent inhibition of microsomally catalyzed covalent binding of acetaminophen by ascorbate has led to the suggestion that ascorbate rapidly reduces the reactive metabolite of acetaminophen and hence should protect against acetaminophen hepatotoxicity. Experimentally, ascorbate given immediately after acetaminophen did not protect against hepatotoxicity in the hamster and did not suppress in vivo covalent binding of acetaminophen. Ascorbate administration resulted in a rapid and marked increase in liver levels of reduced ascorbate. Hepatic ascorbate levels in both control and ascorbate-treated animals were unaffected by the administration of hepatic doses of acetaminophen. Also unchanged by ascorbate in vivo were the overall rate of acetaminophen elimination, acetaminophen-dependent depletion of hepatic glutathione, and the amount of acetaminophen-mercapturate excreted in the urine. Since these indices reflect the hepatic formation and fate of the reactive metabolite of acetaminophen, the data indicate that increased levels of ascorbate did not enhance reduction of the reactive metabolite of the drug back to acetaminophen in vivo, as was suggested from liver microsomal studies. Further studies examined the ascorbate sensitivity of the acetaminophen covalent binding reaction in ascorbate-deficient freshly isolated hepatocytes. In contrast with both the in vitro microsomal and the in vivo binding, covalent binding in the isolated hepatocyte was partially inhibitable by ascorbate. The ascorbate-insensitive binding, which represented approximately 50% of total binding, was inhibitable by cysteine. These data indicate that microsomally catalyzed covalent binding differs significantly from the covalent binding which occurs in the intact liver.

Acetaminophen↗