Comparative analysis of mononuclear cells isolated from mucosal lymphoid follicles of the human ileum and colon.

Studies of human mucosal lymphoid follicles are rare and have been limited to children's Peyer's patches, which are visible at endoscopy. We investigated lymphoid follicles in ileum biopsies of 87 patients and surgical colon specimens from 66 cancer patients, and examined phenotype and function of isolated follicular immune cells. Two (0-10) and 12 (0-117) follicles per patient were found in ileum and colon samples respectively (P < 0.001). The number of lymphoid follicles mononuclear cells (LFMC) that could be isolated per patient was higher from colon compared with ileum specimens [725 000 (0-23 Mio) versus 100 000 (0-1.3 Mio), P < 0.001]. T cells were predominant in both LFMC and lamina propria mononuclear cells (LPMC), but B cells were more and plasma cells less frequent in LFMC. T cells from mucosal follicles were more frequently CD4-positive and CD62L-positive, but less frequently CD8-positive, CD103-positive and CD69-positive than lamina propria T cells. LFMC from ileum compared with colon showed no differences in mononuclear cell composition. Anti-CD3/CD28 stimulation induced similar proliferation of LFMC and LPMC from ileum and colon, as well as secretion of high levels of interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-2, but lower levels of IL-4, IL-6 and IL-10. LFMC from colon secreted more IL-2 than those from ileum. Our study shows that mucosal lymphoid follicles can be identified clearly in adult human colon and yield viable immune cells sufficient for phenotypical and functional analysis. The cellular composition of LFMC from ileum and colon is similar, and both secrete predominantly T helper type 1 cytokines.

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria.

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.

Capsule endoscopy in refractory celiac disease.

BACKGROUND AND STUDY AIMS: Patients with refractory celiac disease (RCD) are at risk of intestinal T-cell lymphoma, which is difficult to diagnose because it often develops in the small bowel. We therefore studied whether wireless capsule endoscopy was able to detect ulcerative jejunitis or intestinal T-cell lymphomas that were missed by standard endoscopic and imaging procedures in patients with RCD. PATIENTS AND METHODS: Detection of ulcerative jejunitis and overt T-cell lymphoma by capsule endoscopy or by upper and lower endoscopy, abdominal computed tomography (CT) or abdominal magnetic resonance tomography (MRT) was compared in 14 consecutive patients with RCD: in seven patients who showed loss of T-cell antigens on intraepithelial lymphocytes and/or clonality of the T-cell receptor gene (i. e. type II RCD) and in seven patients who did not have these features (i. e. type I RCD). RESULTS: Complete evaluation of the small bowel by capsule endoscopy was achieved in 9/14 patients. Signs of ulcerative jejunitis or intestinal T-cell lymphoma, affecting further clinical management, were found in two patients with type II RCD: in one patient these signs were found only by capsule endoscopy (ulcerations and stenosis) and in another patient the abnormalities were identified by CT/MRT (mesenteric lymph nodes harboring lymphoma). No clinically relevant abnormalities were found in patients with type I RCD by lower endoscopy or by small-bowel imaging (capsule endoscopy, CT, or MRT). CONCLUSIONS: In patients with type II RCD, capsule endoscopy can detect additional cases with ulcerative jejunitis and could be included in the diagnostic armamentarium, subject to confirmation by larger series. In patients with type I RCD, our study confirmed the low diagnostic yield of imaging procedures, including wireless capsule endoscopy.

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test.

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.

Susceptibility of different cell layers of the anterior and posterior part of the piriform cortex to electrical stimulation and kindling: comparison with the basolateral amygdala and "area tempestas".

Several lines of evidence suggest that the piriform cortex functions as a generator in the development and propagation of forebrain (limbic type) seizures, particularly in the kindling model of epilepsy. It is, however, not clear where, within the rather large piriform cortex region, the generator resides, and how much tissue is involved. Highly sensitive loci to chemical or electrical stimulation have been described both in the deep anterior and posterior parts of the piriform cortex. Furthermore, data from piriform cortex slice preparations indicated that epileptiform potentials originate in deep structures, particularly the endopiriform nucleus that underlies the piriform cortex. In the present study, in rats, we implanted stimulation and recording electrodes in various rostrocaudal locations of the piriform cortex and endopiriform nucleus, including the "area tempestas", i.e. a structure in the anterior part of the piriform cortex previously proposed to be critically involved in the generation of convulsive seizures of limbic origin. Within the piriform cortex, electrodes were aimed at different cellular layers of this structure. For comparison, additional animals received electrodes in different parts of the basolateral amygdala. A total of 19 different locations was obtained in this way. The susceptibility of these locations to electrical stimulation was characterized by determining the threshold for induction of afterdischarges. The afterdischarge threshold was lowest in layer III of the posterior piriform cortex and some locations in the endopiriform nucleus, whereas amygdala and "area tempestas" displayed higher values. In several animals, particularly those with electrodes in layer III of the posterior piriform cortex, spontaneous spiking was seen in prestimulation recordings, whereas this was never observed in recordings from the amygdala. Subsequent kindling by repeated stimulation of the various locations demonstrated marked differences in afterdischarge threshold reduction and kindling rate. The most marked decreases in afterdischarge threshold were seen in locations within layer III of the piriform cortex, whereas several other locations, including the "area tempestas", exhibited only moderate decreases or no decrease at all. In contrast to previous observations with only few locations in the piriform cortex region, the posterior piriform cortex was not in general slower to kindle than the anterior piriform cortex, although some locations in the posterior piriform cortex exhibited significantly lower kindling rates than the amygdala. The highest kindling rate was seen in the dorsal endopiriform nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of malignant peripheral nerve sheath tumors in European hamsters with 1,1-dimethylhydrazine (UDMH).

A rate of up to 43% of malignant peripheral nerve sheath tumors (PNST) was induced in European hamsters (EH) after weekly s.c. administration of 1,1-dimethylhydrazine (UDMH). The overall neoplastic response in the treated EH was also elevated as compared to the untreated controls. Histologically, the malignant PNST were neurofibrosarcomas and melanotic as well as unpigmented schwannomas. The occurrence of melanotic schwannomas is briefly discussed with regard to the histogenesis of this rare tumor type.

Vinyl chloride: still a cause for concern.

Vinyl chloride (VC) is both a known carcinogen and a regulated chemical, and its production capacity has almost doubled over the last 20 years, currently 27 million tons/year worldwide. According to recent reports it is still a cause for concern. VC has been found as a degradation product of chloroethylene solvents (perchloroethylene and trichloroethylene) and in landfill gas and groundwater at concentrations up to 200 mg/m(3) and 10 mg/L, respectively. Worldwide occupational exposure to VC still seems to be high in some countries (e.g., averages of approximately 1,300 mg/m(3) until 1987 in one factory), and exposure may also be high in others where VC is not regulated. By combining the most relevant epidemiologic studies from several countries, we observed a 5-fold excess of liver cancer, primarily because of a 45-fold excess risk from angiosarcoma of the liver (ASL). The number of ASL cases reported up to the end of 1998 was 197 worldwide. The average latency for ASL is 22 years. Some studies show a small excess risk for hepatocellular carcinoma, and others suggest a possible risk of brain tumors among highly exposed workers. Lung cancer, lymphomas, or leukemia do not seem to be related to VC exposure according to recent results. The mutation spectra observed in rat and human liver tumors (ASL and/or hepatocellular carcinoma) that are associated with exposure to VC are clearly distinct from those observed in sporadic liver tumors or hepatic tumors that are associated with other exposures. In rats, the substitution mutations found at A:T base pairs in the ras and p53 genes are consistent with the promutagenic properties of the DNA adduct 1,N(6)-ethenoadenine formed from VC metabolites. Risk assessments derived from animal studies seem to overestimate the actual risk of cancer when comparing estimated and reported cases of ASL.

Anticonvulsant effects of ipsilateral but not contralateral microinjections of the dopamine D2 agonist LY 171555 into the nucleus accumbens of amygdala-kindled rats.

Recent radioligand binding studies demonstrated an increase in the density of dopamine D2 receptors in the nucleus accumbens ipsilateral to the stimulating electrode in amygdala- or hippocampal-kindled rats. In the present study we examined the anticonvulsant effect of dopamine agonists by unilateral microinjections into the nucleus accumbens in rats kindled from the right basolateral amygdaloid nucleus. Microinjections of the D2 agonist LY 171555 into the ipsilateral nucleus accumbens 15 min prior to the kindling stimulation in fully kindled rats decreased significantly kindling parameters such as seizure severity, seizure duration and afterdischarge duration, whereas the D1 agonist SKF 38393 had no anticonvulsant effects. After ipsilateral microinjection of 40 pmol LY 171555 focal and generalized kindled seizures were totally blocked in almost 50% of the rats. The anticonvulsant effect of LY 171555 could be completely antagonised by systemic administration of the D2 antagonist sulpiride. Microinjection of the D1 or D2 agonist into the nucleus accumbens contralateral to the stimulating electrode had no anticonvulsant effects. In accordance with other reports our data indicate a possible topographic limitation of D2 receptor mediated anticonvulsant effects to specific regions of the basal ganglia.

Structural evidence for the neurotoxicity of methylamphetamine in the frontal cortex of gerbils (Meriones unguiculatus): a light and electron microscopical study.

Morphological alterations in the CNS were investigated in young gerbils treated with two injections of methylamphetamine (Meth) 8 h apart and killed 3, 5 and 7 days after treatment. In gerbils treated with 2 X 35 mg/kg or higher doses of Meth and prepared according to the terminal degeneration method of Gallyas et al. (Stain Technol., 55 (1980) 299-306), silver precipitates occurred in lamina II and III of the frontal cortex. Electron microscopical studies showed degenerated terminals and neurons. After 3 days of survival we found affected but not degenerating pyramidal cells in the same area. Light microscopical observations after 5 and 7 days of survival indicate a recovery of these cells. All described morphological alterations could be suppressed when Meth was administered in combination with haloperidol. These data and the comparison of aminergic projections to the cortex are discussed on the basis of evidence that Meth induces alteration of mesocortical dopamine nerve fibers and their postsynaptic structures in the frontal cortex (FC). Present results indicate that morphological alterations in young gerbils after Meth treatment are limited to the FC. This is in contrast to the literature, which describes neurotoxic effects of Meth in the neostriatum of various adult mammalian species.

The effects of lesions of the posterior piriform cortex on amygdala kindling in the rat.

The piriform cortex (PC) is thought to be critically involved in the genesis of forebrain (limbic type) seizures, including limbic kindled seizures. More recent studies have shown that the posterior PC is particularly sensitive to kindling stimulation, suggesting that the posterior PC contains specific generating sites which may be important for the stepwise progression of kindling. In the present experiments, we used microinjections of ibotenate to study the effect of selective lesions of the posterior PC on amygdala kindling in rats. Large unilateral lesions of the posterior PC and adjacent endopiriform nucleus markedly decreased the susceptibility of the ipsilateral basolateral amygdala to electrical stimulation, thus indicating that the posterior PC may normally contribute to regulation of physiologic excitability in amygdala. During kindling, rats with large lesions of the PC stayed longer in the initial phase of kindling (stage 1) than sham-lesioned controls, consistent with involvement of the posterior PC in the early stages of seizure propagation during kindling acquisition. However, the PC lesions were not capable of blocking or even severely retarding kindling. Following kindling development, rats with large lesions of the posterior PC had significantly higher focal seizure thresholds than kindled rats without lesion or rats with only small PC lesions, which suggests that the posterior PC is involved in the mechanisms which are responsible for the marked increase in seizure susceptibility induced by kindling. Taken together, the data substantiate that PC structures play a facilitatory role in kindling.

Kindling from stimulation of a highly sensitive locus in the posterior part of the piriform cortex. Comparison with amygdala kindling and effects of antiepileptic drugs.

The piriform cortex, especially its deep anterior part, has been recently suggested to be a crucial epileptogenic site in the rat brain. We investigated the susceptibility of different parts of the piriform cortex to the development of electrical kindling as compared to that of the basolateral amygdala. A locus in the deep cell layer (layer III) of the rostral portion of the posterior piriform cortex (PPC) is described, which is considerably more sensitive to electrical stimulation than adjacent areas of the PPC, including the deep prepiriform cortex or the amygdala. The sensitive locus in the PPC can be readily kindled, and focal seizure thresholds in fully kindled rats are 60-90% lower than respective thresholds in rats kindled from other loci. Treatment of fully kindled rats with antiepileptic drugs diazepam, carbamazepine, phenobarbital, and valproate showed that anticonvulsant effects of these drugs in animals kindled from stimulation of the PPC were comparable to respective effects in animals kindled from stimulation of the basolateral amygdala, although the locus in the PPC tended to be more resistant. The data support the idea that the piriform area may contain the most sensitive neuronal tissue responsible for the generation of seizures during kindling. It remains to be determined if the described locus in the PPC is critical to the kindling process when kindling is induced from other structures within the olfactory-limbic system.

Does prolonged implantation of depth electrodes predispose the brain to kindling?

Chronically implanted depth electrodes are widely used for the study of electrical signals generated in deep cerebral locations and for electrical stimulation of such locations. Although the effects of lesions resulting from electrode implantation are generally considered minimal, some reports have shown lasting neurochemical, histological, and behavioral alterations in response to such implantation. Furthermore, there is some evidence that prolonged electrode implantation may decrease the seizure threshold of the implanted region and increases the rate of kindling from this region. This prompted us to undertake a study on different periods of post-surgical delay to onset of electrical stimulation and subsequent characteristics of kindling development. Rats were implanted with a bipolar electrode in the basolateral amygdala, and the threshold for induction of focal paroxysmal activity (afterdischarge threshold, ADT) was determined after post-surgical recovery periods of either 1, 2, 4, or 8 weeks. The animals were then kindled by daily administration of an electrical stimulus until all rats exhibited fully kindled seizures. In fully kindled rats, the ADT was redetermined. Compared to animals with 1 week of electrode implantation, the pre-kindling ADT was significantly lower in rats with 2 and 4 weeks of electrode implantation, but returned towards the 1 week values at 8 weeks. An enhanced kindling rate was seen when kindling stimulations were started after 4 and 8 weeks of electrode implantation. Despite the marked differences in pre-kindling ADT, the post-kindling ADT was similar in the groups with 1, 2, or 4 weeks but significantly lower in the group with 8 weeks post-surgical delay to onset of testing. The data suggest that prolonged implantation of a bipolar electrode into a sensitive region of the limbic system predisposes the brain to kindling. Based on previous observation of iron deposits induced by electrode implantation and the epileptogenic effect of iron in cortical and limbic regions, we propose that the present observations are due to deposition of iron from hemoglobin destruction in local microhemorrhages caused by the implantation.

Effect of selective bilateral destruction of the substantia nigra on antiepileptic drug actions in kindled rats.

The substantia nigra (SN) is thought to be involved in the regulation of seizure activity and there is evidence that the nigra might be a site of anticonvulsant drug action, especially in the case of drugs that act by potentiating gamma-aminobutyric acid (GABA)-mediated neurotransmission. The current studies monitored the anticonvulsant effect of four major antiepileptic drugs, i.e. valproic acid, carbamazepine, phenobarbital and diazepam, in fully kindled rats before and after bilateral destruction of the SN. Rats were kindled by stimulation of either the basolateral amygdala or the piriform cortex. The SN was lesioned bilaterally by microinjection of ibotenic acid, and only animals with near-complete, selective destruction of the SN were used for final evaluation of the anticonvulsant drug experiments. The behavioural characteristics and the duration of fully kindled seizures were not altered by bilateral destruction of the SN. Phenobarbital, diazepam and valproate significantly reduced kindled seizures severity and duration before and after the SN lesions. Carbamazepine was the only drug to show a marked decrease in its anticonvulsant effects after SN lesion. Since benzodiazepines, valproate and phenobarbital are though to enhance GABAergic transmission, the lack of effect of SN lesions on the anticonvulsant effects of these drugs argues against the suggestion that the SN is the anatomical site responsible for exerting anticonvulsant effects in response to drug-induced augmentation of GABA transmission.

Electron microscopical evidence for common inner ear and lateral line efferents in urodeles.

The efferents of the lateral line system and the inner ear were examined in urodeles using retrograde labelling with horseradish peroxidase (HRP). The Golgi-like filling thus achieved allowed tracing of the axons of efferent cells from one inner ear to the other and from the lateral line nerves into both inner ears. Electron microscopic examination of the inner ear revealed HRP label only in vesicle filled terminals on hair cells traditionally considered as efferent synapses. These data confirm earlier claims of common bilateral inner ear and lateral line efferents. In addition, the efferent nature of retrogradely labelled rhombencephalic cells is proven by their continuity with ultrastructurally identified efferent synapses in the inner ear.

Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats.

Enhancement of GABAergic transmission in the substantia nigra has been shown to attenuate motor manifestations of diverse seizure models, including kindling. Similar anticonvulsant effects were reported after bilateral lesions of the substantia nigra, supporting the view that the nigra efferents constitute a critical gating mechanism in the propagation of seizure activity. However, in the lesion studies reported so far the nigra was not destroyed selectively so that regions destroyed in addition to the nigra could have been involved in the anticonvulsant effects observed. We destroyed the nigra selectively in fully kindled rats by bilateral microinjection of small amounts of the neurotoxin ibotenic acid. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the piriform cortex. In both groups, there was no indication of a reduction in seizure susceptibility, seizure severity or seizure duration after bilateral destruction of the nigra. The data thus indicate that, at least in kindled rats, the substantia nigra might be less important for seizure generation and/or propagation than previously thought.

Lesions of the deep prepiriform cortex ('area tempestas') in rats do not affect the convulsant action of systemically administered bicuculline.

The gamma-aminobutyric acid (GABA) antagonist, bicuculline, induces generalized motor seizures when injected into a discrete site ('area tempestas') in the deep prepiriform cortex at concentrations considerably lower than those that induce convulsions from closely adjacent areas or other forebrain sites such as amygdala and hippocampus. This observation prompted the suggestion that the area tempestas is a crucial epileptogenic site involved in seizure generation. In the present study, the region functionally defined as area tempestas in rats was bilaterally destroyed by microinjection of ibotenic acid. Systemic administration of bicuculline induced generalized motor seizures in both lesioned and non-lesioned animals without any indication of differences in seizure severity or latency. This argues against the suggestion that the deep prepiriform cortex plays a crucial role in the generation of seizures following systemic administration of GABA antagonists.

Lack of changes in seizure susceptibility during the estrous cycle in kindled rats.

The threshold and pattern of focal and generalized seizures in fully kindled rats during the estrous cycle were investigated. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the anterior portion of the posterior piriform cortex. Determinations of the threshold for focal afterdischarges were either carried out in the morning (between 8 and 9 a.m.) or in the afternoon (between 2 and 3 p.m.). In all experiments, stable and reproducible afterdischarge thresholds were obtained during the different stages of the estrous cycle. The only significant alteration was a reduced seizure duration during metestrus and/or the first day of diestrus in amygdala kindled rats. The data indicate that the natural changes in sex hormone levels during the estrous cycle in rats do not affect seizure susceptibility, at least in the kindling model of epilepsy.

Effects of valproate and E-2-en-valproate on functional and morphological parameters of rat liver. III. Influence of fasting.

Valproate (VPA) therapy has been associated with a rare but fatal hepatotoxicity. Several possible biochemical mechanisms responsible for the hepatotoxicity have been proposed, but the matter has not been decided. There is some evidence that VPA-associated hepatotoxicity may represent the consequences of a VPA overload on a limited mitochondrial beta-oxidation capacity, causing abnormalities in metabolic pathways. If this assumption is true, fasting-induced increase of endogenous fatty acids, which compete with VPA for beta-oxidation, should enhance the hepatotoxic potential of VPA. Indeed, involuntary fasting because of anorexia, e.g., in children with febrile infections, has been discussed as one clinical risk pattern preceding VPA-associated hepatic fatalities. In the present experiments, the effects of fasting on functional and morphological parameters of the liver were studied in young male rats chronically treated with VPA. E-2-en-VPA (trans-2-en-VPA), a major active metabolite of the beta-oxidation pathway of VPA, was used for comparison. Both drugs were administered at doses of 250 mg/kg i.p. 3 times daily for 1 week. In control rats, a 40-h fasting period resulted in marked mobilization of liver lipid and glycogen stores, alterations in liver enzyme activities, and hyperammonemia. In rats treated with VPA, fasting reduced beta-oxidation of the drug, but seemed not to increase its hepatotoxic potential. Compared to experiments without fasting, alterations in liver enzymes and ammonia levels induced by VPA were less marked or absent in fasted rats, and histopathological examination of liver sections did not indicate degenerative liver lesions in response to drug treatment. Thus, compared to previous rat studies on VPA without fasting, fasting appeared to attenuate VPA's hepatotoxic potency, possibly as a result of fasting-induced increases in carnitine levels. In rats treated with E-2-en-VPA, no indication of hepatotoxicity was evident, and alterations in functional hepatic parameters were less pronounced than with VPA. The data do not indicate that fasting or poor nutrition are risk factors for VPA-associated hepatic injury.

Effects of valproate and E-2-en-valproate on functional and morphological parameters of rat liver. I. Biochemical, histopathological and pharmacokinetic studies.

E-2-en-Valproate (E-2-en-VPA; trans-2-en-VPA) and VPA were studied for potential hepatotoxicity in young male Sprague-Dawley rats. Both drugs were administered daily at 750 mg/kg i.p. (divided into three doses a day) for 7 consecutive days. Clinical chemistry parameters were studied before and after the period of treatment. Furthermore, the drug pharmacokinetics and metabolism were analyzed at onset and end of the prolonged administration. Treatment with VPA induced hyperammonemia and other alterations in liver function tests which were not observed after treatment with E-2-en-VPA, although plasma levels of both drugs were comparable. The pharmacokinetics of VPA and E-2-en-VPA in young rats were similar, but analysis of metabolites by gas chromatography-mass spectrometry indicated marked differences in the metabolite profile, e.g., a lack of the suspected hepatotoxic metabolite 4-en-VPA in plasma of rats treated with E-2-en-VPA. Histopathological examination of liver sections showed that VPA and E-2-en-VPA did not induce degenerative liver lesions or significant alterations in hepatic content and distribution of lipids and glycogen at the doses administered. Only one of the VPA treated rats showed fatty infiltration (microvesicular steatosis). The data demonstrate that, although E-2-en-VPA is more potent than VPA as an anticonvulsant in rats, it does not exert more potent hepatotoxic effects and does not alter ammonia metabolism. Thus the data substantiate previous experimental studies that E-2-en-VPA might be a valuable substitute for VPA.

Effects of valproate and E-2-en-valproate on functional and morphological parameters of rat liver. II. Influence of phenobarbital comedication.

The effect of phenobarbital on the potential hepatotoxicity of E-2-en-valproate (E-2-en-VPA; trans-2-en-VPA) and VPA was studied in young male Sprague-Dawley rats. E-2-en-VPA and VPA were administered daily at 750 mg/kg i.p. (divided into three doses a day) for 7 consecutive days. Phenobarbital was coadministered i.p. once daily at 100 mg/kg for 2 days, followed by daily injections of 50 mg/kg for the subsequent days of the treatment period. Additional groups of rats were treated with phenobarbital alone or received once daily administration of 4-en-VPA (100 mg/kg), a potentially hepatotoxic metabolite of VPA. Clinical chemistry data were studied before and after the period of treatment. Furthermore, drug and metabolite levels were analyzed by gas chromatography-mass spectrometry. Treatment with VPA and phenobarbital resulted in deaths and histopathological liver alterations, such as marked microvesicular steatosis and degenerative lesions, whereas no death and hepatotoxicity occurred in rats treated with E-2-en-VPA and phenobarbital. Furthermore, hyperammonemia was recorded in VPA- but not E-2-en-VPA-treated rats. In comparison to treatment with VPA or E-2-en-VPA alone, combined treatment with phenobarbital markedly reduced plasma levels of the parent drugs and metabolites originating from beta-oxidation, but, in case of VPA, increased metabolites originating from omega-oxidation. Plasma levels of 4-en-VPA were increased by phenobarbital in VPA-treated rats, but 4-en-VPA was not detectable in rats treated with E-2-en-VPA. The most severe alterations in functional and morphological liver parameters were found in rats treated with 4-en-VPA. In these animals, the extent of steatosis was significantly correlated with plasma levels of 4-en-VPA, but not its major metabolite 2,4-dien-VPA. Plasma levels of 4-en-VPA or its major metabolite 2,4-dien-VPA in rats without steatosis were markedly higher than levels of these compounds in VPA-treated rats with steatosis, suggesting that 4-en-VPA and 2,4-dien-VPA are not critically involved in the hepatotoxic effects of VPA. The data substantiate that E-2-en-VPA is less hepatotoxic than VPA and may thus offer advantages for antiepileptic therapy.