Inhibition of testicular testosterone biosynthesis following experimental varicocele in rats

In an attempt to determine whether defective testicular testosterone (T) biosynthesis may be associated with a varicocele, an experimental study was performed in adult rats whereby a unilateral left varicocele was surgically created. At 2, 4, 8, and 12 wk following the creation of the varicocele, intratesticular T as well as the activities of three (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) of the five enzymes in the delta 4 pathway of testicular T biosynthesis were measured. Intratesticular T (ng/g testis +/- SEM) in the left testis decreased significantly from 121 +/- 21 in the control group to 59 +/- 8 in the two-wk varicocele group (p less than 0.01), and remained significantly suppressed throughout the experimental period. The T concentrations in the right testis paralleled those in the left in both the control and varicocele animals. At 2 wk following the creation of the varicocele, the activity (nmol/min/testis +/- SEM) of the 17,20-desmolase enzyme decreased significantly, from 115 +/- 8 in the left testis of control rats to 87 +/- 6 in the left testis of the varicocele animals (p less than 0.025), and remained low throughout the 12 weeks of the study. The activity of the 17 alpha-hydroxylase enzyme was significantly decreased at the 8th and 12th weeks of the study, while the 17 beta-hydroxysteroid dehydrogenase activity did not show any significant change during the study period. The enzyme activities in the right testis paralleled those in the left testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Effects of ethanol and acetaldehyde on the biosynthesis of testosterone in the rodent testes

The effects of ethanol and acetaldehyde on testicular steroidogenesis were examined in enzymatically dispersed cells of the rodent testes. Both drugs significantly inhibited gonadotropin-stimulated steroidogenesis, but acetaldehyde was considerably more potent (>1000 times) than ethanol. To determine the step in testosterone's biosynthetic pathway which was inhibited by the two drugs, cells were incubated in the presence of [³H]pregnenolone and [³H]progesterone, and the amount of label incorporated into testosterone and its precursors was determined. Ethanol and acetaldehyde inhibited only the conversion of androstenedione to testosterone; none of the other precursors of testosterone was affected.     

Inhibition of testosterone biosynthesis by ethanol: multiple sites and mechanisms in dispersed Leydig cells

Isolated rat Leydig cells were incubated for 2 h in sealed polycarbonate tubes under O2/CO2 atmosphere with 10 mIU/ml human chorionic gonadotropin. 20 mmol/l ethanol reduced the concentration of testosterone (16%, P less than 0.025); raised the concentrations of pregnenolone (60%, P less than 0.001), androstenedione (86%, P less than 0.001) and dehydroepiandrosterone (81%, P less than 0.001); but did not change concentrations of progesterone and 17 alpha-hydroxyprogesterone in the incubation medium. Ethanol also raised the lactate/pyruvate ratio in the Leydig cell suspension. 4-Methylpyrazole (0.5 mmol/l) abolished the ethanol-induced changes. The present results suggest that ethanol inhibits testosterone synthesis in isolated rat Leydig cells at the pregnenolone-to-testosterone pathway by inhibiting 3 beta-hydroxy-5-ene-steroid dehydrogenase/5-ene-4-ene-isomerase catalyzed reactions and the conversion of androstenedione to testosterone. These inhibitions are caused by consequences of ethanol metabolism. A likely mechanism for the former inhibition is that the increase in the NADH/NAD+ ratio in Leydig cells leads to inhibition of reactions catalyzed by 3 beta-hydroxy-5-ene-steroid dehydrogenase/5-ene-4-ene isomerase, but the inhibition mechanism operating at the androstenedione-to-testosterone step remains to be characterized.     

Inhibition of acetaminophen activation by ethanol and acetaldehyde in liver microsomes

Mechanisms of the inhibitory effect of ethanol on acetaminophen hepatotoxicity are controversial. We studied the effects of ethanol and acetaldehyde, an oxidative metabolite of ethanol, on NADPH-dependent acetaminophen-glutathione conjugate production in liver microsomes. Ethanol at concentrations as low as 2mM prevented the conjugate production noncompetitively. Acetaldehyde also inhibited acetaminophen-glutathione conjugate production at concentrations as low as 0.1mM that is comparable with those observed in vivo after social drinking. Acetaldehyde may be involved in ethanol-induced inhibition of acetaminophen hepatotoxicity.     

Inhibition of testosterone secretion by digitoxin in rat testicular interstitial cells.

Both in vivo and in vitro experiments were conducted to determined the effects of digitoxin on the secretion of testosterone, and its underlying mechanisms including testicular adenosine 3':5'-cyclic monophosphate (cAMP), and the activities of steroidogenic enzymes. Male rats were injected with digitoxin, human chorionic gonadotropin (hCG), or hCG plus digitoxin via a jugular catheter. Blood samples were collected immediately before and at 30 and 60 min after the challenge, and analyzed for testosterone by radioimmunoassay. In an in vitro study, rat testicular interstitial cells were isolated and incubated with digitoxin, hCG, 8-bromo-cAMP (8-Br-cAMP), digitoxin plus hCG, or digitoxin plus 8-Br-cAMP at 34 degrees C for 1 h. The media were collected and analyzed for testosterone. For studying cAMP accumulation, testicular interstitial cells were incubated for 1 h in the medium containing isobutyl-1-methylxanthine (IBMX) and different doses of digitoxin with the absence or presence of hCG. After incubation, cells were processed for determining cAMP content. Intravenous injection of digitoxin decreased hCG-stimulated, but not basal, plasma testosterone levels. Administration of digitoxin in vitro resulted in an inhibition of both basal and hCG- as well as 8-Br-cAMP-stimulated release of testosterone. In addition, digitoxin diminished hCG-stimulated cAMP accumulation in rat testicular interstitial cells. Furthermore, digitoxin inhibited the activity of cytochrome P450 side chain cleavage enzyme (P450scc) but failed to affect the activities of other steroidogenic enzymes. Taken together, these results suggest that the acute inhibitory effect of digitoxin on the testosterone production in testicular interstitial cells involves, at least partly, an inefficiency of post-cAMP events, and a decrease of P450scc activity.     

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.     

Inhibition of testosterone secretion by S-petasin in rat testicular interstitial cells

S-petasin, a kind of sesquiterpene ester, is the anti-inflammatory ann analgesic component of the butterbur (Petasites hybridus). The clinical benefit of S-petasin is the spasmolytic activity, but its side effects on the reproductive endocrinology are not clear yet. The present study was to explore the effects of S-petasin on the secretion of testosterone in vivo and in vitro. We found that single intravenous injection of S-petasin (1 microg/kg) decreased basal plasma testosterone concentration in adult male rats. The enzymatically dispersed rat testicular interstitial cells were incubated with S-petasin (0-4.3 x 10(-5)M) in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (adenylyl cyclase activator, 10(-5) M), and androstenedione (testosterone biosynthesis precursor, 10(-9) M) at 34 degrees C for 1 h. The concentrations of testosterone in the incubation medium were measured by radioimmunoassay. S-petasin at 4.3 x 10(-7) M was effective to reduce the basal and hCG-stimulated release of testosterone in rat testicular interstitial cells. The stimulatory effects of testosterone secretion induced by forskolin and androstenedione were significantly reduced by S-petasin at 4.3 x 10(-5) M and 4.3 x 10(-6) M, respectively. These results suggest that S-petasin inhibits the production of testosterone in rat testicular interstitial cells in part through diminishing the activities of adenylyl cyclase and 17-ketosteroid reductase.     

Inhibition of testicular androgenesis by urinary antigonadotropins and melatonin in rats.

Comparative in vitro and in vivo studies of the effects of urinary gonadotropin-inhibiting substances and melatonin on the androgenesis in rat testicular homogenates were performed. When the urinary extract containing the inhibiting substances or melatonin was added directly to the incubation medium, and was also injected into rats 24 and 48 hrs prior to sacrifice, either one was effective in suppressing the conversion of pregnenolone to testosterone and/or androstenedione in testicular tissues. The urinary extract exerted the inhibitory effect on the conversion of cholesterol to pregnenolone in vitro and in vivo, whereas melatonin did not have this effect in vitro and in vivo. These findings suggest that the antigonadotropic substances are different from melatonin in their action on androgenesis in the rat testes.     

Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: central role of mitochondrial NADH redox state

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonadotropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by L-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.     

Concomitant increase in hepatic triacylglycerol biosynthesis and cytosolic fatty-acid-binding-protein content after feeding rats with a cholestyramine-containing diet.

Cholestyramine feeding of rats increased the rate of palmitate and glycerol incorporation into triacylglycerols of isolated hepatocytes. Concomitantly an increase of fatty-acid binding by hepatic cytosolic proteins was observed, which could be attributed to an elevation of the content of the fatty-acid-binding protein (Mr 12000). The involvement of this protein in cholesterol, bile-acid and triacylglycerol metabolism is discussed.     

Reduction of acetaldehyde to ethanol by some micro-organisms and its stereospecificity.

The stereochemical course of the reduction of acetaldehyde to ethanol was investigated by evaluating, with the enzymic system yeast alcohol dehydrogenase/diaphorase and g.c.-m.s., the configuration of [1-2H]ethanol obtained from [1-2H]acetaldehyde with different micro-organisms. Although only S-[1-2H]ethanol was formed, all the micro-organisms showed evidence of the existence of alcohol dehydrogenases with opposite stereospecificity.     

Effect of sub-chronic endosulfan exposures on plasma gonadotrophins, testosterone, testicular testosterone and enzymes of androgen biosynthesis in rat

Insecticide endosulfan significantly inhibited testicular androgen biosynthesis in adult rats, when fed (po) at 7.5 and 10 mg/kg body weight dose levels, consecutively for 15 and 30 days. No appreciable alterations were apparent in body weights, testicular wet weights, and cytosolic and microsomal protein contents of testis in treated rats. Profound decrease in the levels of plasma gonadotrophins (FSH and LH) along with plasma testosterone and testicular testosterone were observed at both the doses of endosulfan, particularly after the longer exposure of 30 days. Activities of steroidogenic enzymes studied (3 beta- and 17 beta-hydroxysteroid dehydrogenases) were considerably lowered on longer exposure of endosulfan. A significant decrease in the contents/activities of microsomal cytochrome P-450 and related mixed function oxidases (MFOs) in testis of treated rats was also observed, along with a marked inhibition in the activity of cytosolic conjugation enzyme, glutathione-S-transferase at both doses studied. These biochemical changes were reversed when the endosulfan treatment was withdrawn.     

The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism

The effects of ketone body metabolism suggests that mild ketosis may offer therapeutic potential in a variety of different common and rare disease states. These inferences follow directly from the metabolic effects of ketosis and the higher inherent energy present in d-beta-hydroxybutyrate relative to pyruvate, the normal mitochondrial fuel produced by glycolysis leading to an increase in the DeltaG' of ATP hydrolysis. The large categories of disease for which ketones may have therapeutic effects are:(1)diseases of substrate insufficiency or insulin resistance,(2)diseases resulting from free radical damage,(3)disease resulting from hypoxia. Current ketogenic diets are all characterized by elevations of free fatty acids, which may lead to metabolic inefficiency by activation of the PPAR system and its associated uncoupling mitochondrial uncoupling proteins. New diets comprised of ketone bodies themselves or their esters may obviate this present difficulty.     

The effects of ketone bodies,bicarbonate, and calcium on hepatic mitochondrial ketogenesis

The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 mm calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH₂ via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD⁺ from NADH₂ during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.     

Role of cytosolic rat liver aldehyde dehydrogenase in the oxidation of acetaldehyde during ethanol metabolism in vivo.

The activity of a high-Km aldehyde dehydrogenase in the liver cytosol was increased by phenobarbital induction. No corresponding increase in the oxidation rate of acetaldehyde in vivo was found, and it is concluded that cytosolic aldehyde dehydrogenase plays only a minor role in the oxidation of acetaldehyde during ethanol metabolism.     

Determination of metabolite compartition in hepatic cells by varying the redox state in vivo.

In the present study, metabolite (lactate, pyruvate, glycerol 3-phosphate, dihydroxyacetone phosphate) concentrations were measured in various redox states. The mathematical relations between metabolite concentrations in various redox states were expressed algebraically and studied. Models which provided separate lactate/pyruvate (L/P) and glycerol 3-phosphate/dihydroxyacetone phosphate (G/D) spaces correspond to the experimental results in the case of "reductants" (e.g. ethanol, acetaldehyde, dihydroxyacetone and acetate) or of "oxidizing agents" (e.g. pyruvate) of the cytosolic NAD-NADH. Crotonate injection caused an oxidation of cytosolic redox couples, but no separation of the lactate/pyruvate space from the glycerol 3-phosphate/dihydroxyacetone phosphate space may necessarily be inferred. Furthermore, the following statements could be made in both first cases: (i.e. of "reductants" and "oxidizing agents"): (a) Redox couples in L/P space and in G/D space (together L/P-G/D system) are in equilibrium; (b) Redox-equivalent transport from the L/P space to the G/D space is not subject to any velocity-limiting mechanism; (c) Substrates which transports redox-equivalents into and from the L/P-G/D system reach concentrations to values, which are in a linear relation to each other in this system; (d) It is possible that these substrates are regenerated in another system which is also in equilibrium and subject to statement c.