Leon Lack

Neurotoxicity of glycidamide, an acrylamide metabolite, following intraperitoneal injections in rats

Acrylamide (2-propenamide) monomer produces central-peripheral distal axonopathy in humans and some animal species. Its neurotoxicity is characterized by abnormal sensation, decreased motor strength, and ataxia. Acrylamide forms adducts with glutathione, proteins, and DNA. Recent studies demonstrated that acrylamide is metabolized to its epoxide, glycidamide (2,3-epoxy-1-propanamide). We studied the neurotoxicity potential of glycidamide in male Sprague-Dawley rats. Animals (groups of 6) were injected ip daily with either aqueous acrylamide or glycidamide at an acrylamide-equivalent dose of 50 mg/kg (0.70 mmol/kg). Both treatments resulted initially in the rats circling, which was followed by the onset of ataxia at 7-9 d and hindlimb paralysis at 12-14 d. Treated animals showed muscle wasting. At termination, acrylamide- and glycidamide-treated rats weighed 105% and 86% of initial weight, respectively, compared to 145% for controls. Animals were anesthetized and perfused with 10% neutral phosphate-buffered formalin 12 or 14 d after beginning of treatment. Both treatment groups exhibited similar neuropathologic changes in the central and peripheral nervous systems. More severe lesions were produced by glycidamide. A marked increase in the number of affected Purkinje cells in the cerebellum, which exhibited changes ranging from pyknosis to cell death, were present. The brainstem exhibited axonal degeneration with chromatolytic necrosis in midbrain medial and lateral reticular nuclei. The spinal cord was characterized by spongy form changes with vacuoles of different sizes in various levels. These results suggest that glycidamide is an active neurotoxic metabolite of acrylamide.

Taurocholate uptake by membrane vesicles prepared from ileal brush borders

Abstract Taurocholate uptake by vesicles prepared from brush borders obtained from the small intestines of guinea pigs was studied. Vesicles obtained from the brush borders of ileums demonstrated an enhanced initial uptake in those incubations where a sodium ion gradient (extravesicular sodium concentration greater than intravesicular) was present at the outset. With the dissipation of this sodium gradient the intravesicular concentration of taurocholate declined. This overshoot phenomenon was absent in parallel incubations of vesicles made from jejunal tissue. When the sodium chloride was replaced by isosmotic amounts of mannitol no overshoot was observed in incubations of ileal vesicles until subsequent addition of sodium chloride to these incubations. These observations are in accord with the idea that those subcellular structural elements operating in the ileal bile salt transport system are associated with the brush border membranes of the ileal mucosal cells.

Prostatic cancer. I. 6-Methylene-4-pregnen-3-ones as irreversible inhibitors of rat prostatic Δ4-3 ketosteroid 5α-reductase☆

Abstract Some derivatives of 6-methylene-4-pregnen-3-one were studied as inhibitors of Δ4-3-ketosteroid 5α-reductase. Maximum inhibitory activity was shown by 17-acetoxy-6-methylene-4-pregnene-3,20-dione (AMPD). Irreversible inactivation was observed following preincubation of the enzyme with NADPH and AMPD. This inactivation was found to occur only in the presence of NADPH. As such enzyme inactivation was not due to the formation of a more inhibitory metabolic product, or to the formation of Superoxide via a cytochrome P-450/NADPH pathway, it seemed likely that the observed inactivation was derived from an irreversible combination of the enzyme with AMPD. That this was probably the case was established by kinetic studies which revealed a pattern compatible with a kcat type of mechanism.

Prostatic cancer—II. Inhibitors of rat prostatic 4-ene-3-ketosteroid 5α-reductase derived from 6-methylene-4-androsten-3-ones

The studied 6-methylene-4-androsten-3-ones proved to be significantly inferior to 6-methylene-4-pregnene-3,20-dione and its 17-acetoxy derivative described in Part 1 as inhibitors of 4-ene-3-ketosteroid 5 alpha-reductase [1] in vitro. Surprisingly, the 6-methylene derivative of testosterone was only weakly active until acetylated, when an effective inhibitor was obtained. Etherification of the hydroxyl-group, its replacement by a hydrocarbon chain, or introduction of a substituent at C17 or on the methylene group led to virtual loss of activity. 17 alpha-Chloro-6-methylene-4-androstene-3-one had ca 60-70% of the potency of progesterone, but was inactive as enzyme inhibitor in explants of rat prostate in tissue culture and in in vivo studies. 6-Methylenetestosterone acetate was weakly active as enzyme inhibitor in explants of human prostate in tissue culture and produced a histological picture closely resembling testosterone and differing from that of cyproterone acetate. In vivo in the rat it had 80% of the androgenic activity of testosterone propionate. The foregoing data have been used to define some structural characteristics necessary for enzyme inhibition and to draw some conclusions regarding the architecture of the androgen and progesterone receptors and of the enzyme active site.

Ion requirements for taurocholate transport by ileal brush border membrane vesicles

Abstract The ion requirements for intestinal taurocholate transport were studied using vesicles prepared from the brush borders of guinea pig small intestines. For each experimental electrolyte, parallel uptake experiments were performed with vesicles from jejunal and ileal brush border membranes to differentiate between uptake by passive fluxes and non-specific binding and uptake by the ileal bile salt active transport system. Uptake of taurocholate prior to the addition of electrolyte was the same for vesicles prepared from jejunal and ileal tissue. During the presence of a sodium gradient (extravesicular concentration greater than intravesicular), only ileal vesicles displayed the enhanced uptake which is characteristic of the overshoot phenomenon. When NaCl was replaced by KCl or LiCl, the overshoot was not observed. Replacement of NaCl with NaCNS, Na 2 SO 4 , or NaSO 3 C 2 H 4 OH, however, resulted in no significant difference in the initial uptake values observed in either the jejunal or ileal vesicles. This pattern of taurocholate transport independence of relative anion permeability differs from the pattern observed by others for the Na + dependent transport of D-glucose by intestinal brush border membrane vesicles. This difference may be attributed in part to the fact that, unlike the situation with glucose, the binding of a taurocholate anion and a sodium cation by the hypothetical carrier would result in an electroneutral addition.

Inhibitory effects of some steroidal 6-methylene derivatives on 5α-reductase activity in human and rat prostate

Using a short-term organ culture assay, some 6-methylene derivatives of progesterone and testosterone have been evaluated for their effects on testosterone metabolism in rat and human prostatic tissues, and on DNA synthesis in explants from 7-day castrated rats. Comparative studies showed that the ability to inhibit 5 alpha-reductase activity was fairly specific with respect to structural requirements. Methylene substitution at the C6 position of the progesterone molecule was associated with high inhibitory activity. In explants prepared from human prostates, 6-methylene progesterone (II) had 70-85% (mean of 79% for 4 BPH tissues) of the potency of unmodified progesterone (I). Its 17 alpha-acetoxy-6-methylene analog (III), however, had only 32-73% (mean of 53% for 5 BPH specimens) of the activity of (I). The degrees of inhibition in rat and human prostatic tissues were similar. Inhibition of 5 alpha-reductase activity in cultured explants by 6-methylene progesterone (II) could not be reversed by change in media. The 6-methylene derivatives had little or no effect on DNA synthesis. Histological examination confirmed a lack of effect on basal cell proliferation. However, morphological alterations affecting epithelial cell height and secretory activity were clearly evident. These results indicate that, under our experimental conditions, the main effect of inhibition of 5 alpha-reductase activity in prostatic tissues by 6-methylene derivatives of progesterone is related to suppression of differentiated function.

Bile Salt Transport Systems

There are three sites where the active transport of bile salts has been described. Two of these, the liver and the ileum, are integral parts of the enterohepatic circulation. The third site is the proximal renal tubule, which can reabsorb bile salts from the glomerular filtrate in those situations where appreciable concentrations of bile salts exist in the peripheral (i.e., non-portal) plasma. In addition to these presumed active transport sites, passive transepithelial movement of bile salts has been described in various regions of the gastrointestinal tract. These passive processes play a recognizable role in the enterohepatic circulation of the total bile salt pool, but the quantitative contribution of these passive processes is not precisely known. A number of fairly extensive reviews on the transport of bile salts and related substances have appeared (1–3).

Binding of sodium taurocholate by bovine serum albumin

Abstract Studies employing equilibrium dialysis methods were made of the binding of sodium taurocholate by bovine serum albumin. In the absence of fatty acids a maximum of one (1.01 ± 31) mole of sodium taurocholate is bound per mole of albumin. The dissociation constant was found to be 8.21 · 10−5 ± 0.26 · 10−5 M. The presence of 1 · 10−4 M sodium oleate increases the maximum stoichiometry of binding to 3 moles of bile salt per mole of bovine serum albumin. However, the fatty acid did not appreciably alter the dissociation constant.

The effect of bile salts on the formation and hydrolysis of cholesterol esters by rat liver enzymes

To determine the effects of different bile salts on the enzymic esterification of cholesterol and the hydrolysis of cholesterol esters rat liver homogenates and rat liver microsomes were incubated with varying amounts of different bile salts. Bile salts inhibited the formation of radioactive cholesterol esters in incubations of either rat liver homogenates or rat liver microsomes containing [14C]cholesterol. Chenodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate were more potent inhibitors than their comparable cholate analogues. Bile salts stimulated the hydrolysis of cholesterol esters when incubation were carried out with the liver homogenates. The dihydroxy bile salts were again more potent in this regard than the trihydroxylated bile salts. When the effects of bile salts on cholesterol ester hydrolysis were studied in in vitro incubations of hepatic microsomes a biphasic mode of acion was observed. In the absence of Na+ or K+ bile salts stimulated the hydrolysis of cholesterol oleate. However, following the addition of either Na+ or K+ to the microsomal incubations, bile salts caused an inhibition of cholesterol ester hydrolysis. Since cholesterol esterification was also inhibited under these conditions a direct inhibitory effect (not attributable to enhanced hydrolase activity) of the bile salts on the formation of cholesterol esters by the microsomes was established. Furthermore, this inhibition takes place at the transacylation step involving the fatty acyl-CoA ester and the sterol. These results suggest that bile salts can significantly alter the cholesterol-cholesterol ester profile in the liver, and furthermore, that these effects may be influenced by small changes in the intracellular environment in the region where these reactions occur.

Protein‐bound pyrroles in rat hair following subchronic intraperitoneal injections of 2,5‐hexanedione

Studies were initiated to ascertain whether body hair could be used to develop a biological marker for chronic exposure to industrial neurotoxicants that yield the metabolite 2,5-hexanedione (2,5-HD), that is, n-hexane and methyl n-butyl ketone. Rats were injected daily with a 50 mg/kg ip dose of 2,5-HD for 45 d. At intervals, body hair and individual vibrissae were removed (under general anesthesia) and tested for the presence of pyrrole substances with p-N,N-dimethylaminobenzaldehyde (DMAB, Ehrlichs reagent). Vibrissae and body hair were stained a reddish color that was distinctly different from that observed with the hair taken from control animals. Solubilized body hair protein from the treated animals gave a positive Ehrlichs test, while that from control animals was negative. Spectral analysis of the DMAB-treated hair from experimental animals disclosed a maximum absorbance at 530 nm, which indicated the presence of pyrrole substituents. Serial analysis of individual nose hairs taken during 2,5-HD administration showed a progression with time of the region staining positively for pyrroles, thus indicating that the process can proceed in growing hair. These findings suggest the potential utility of hair as an indicator for chronic exposure to this class of industrial chemicals possessing neurotoxicity potential. This could complement urinary analysis, which is now used to confirm recent exposure.