Cytisine inhibits the protective activity of various classical and novel antiepileptic drugs against 6 Hz-induced psychomotor seizures in mice.

BACKGROUND: Cytisine (CYT) is a partial agonist of brain α4ß2 nicotinic acetylcholine receptors widely used in Central/Eastern Europe for smoking cessation. OBJECTIVES: This study evaluated the effect of CYT on the ability of classical and novel antiepileptic drugs to prevent seizures evoked by the 6-Hz test, a model of psychomotor seizures in mice thought as a model of drug-resistant seizures. RESULTS: CYT administered intraperitoneally (i.p.) in a dose of 2 mg kg-1 significantly inhibited the anticonvulsant activity of lacosamide, levetiracetam, and pregabalin, increasing their median effective doses 50 (ED50) values from 6.88 to 10.52 mg kg-1 (P < 0.05) for lacosamide, from 22.08 to 38.26 mg kg-1 (P < 0.05) for levetiracetam, and from 40.48 to 64.61 mg kg-1 (P < 0.01) for pregabalin, respectively. There were no significant changes in total brain concentrations of lacosamide, levetiracetam, and pregabalin following CYT i.p. administration. CYT administered in a dose of 2 mg kg-1 failed to change the protective action of clobazam, clonazepam, phenobarbital, tiagabine, and valproate in the 6-Hz test. Neither CYT (2 mg kg-1) alone nor its combination with the anticonvulsant drugs (at their ED50 values from the 6-Hz test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; and grip strength and passive avoidance tests, respectively. CONCLUSION: CYT-evoked alterations in the protection provided by some antiepileptic drugs against seizures can be of serious concern for epileptic smokers, who might demonstrate therapeutic failure to lacosamide, levetiracetam, and pregabalin, resulting in possible breakthrough seizure attacks.

Effects of gamma-decanolactone on seizures induced by PTZ-kindling in mice.

Gamma-decanolactone is a monoterpene compound, and its psychopharmacological evaluation in mice revealed that it has a dose-dependent effect on the central nervous system, with hypnotic, anticonvulsant, and hypothermic activity. The aim of the present study was to investigate the effect of gamma-decanolactone on pentylenetetrazole (PTZ)-kindling in mice. Phenobarbital, an antiepileptic drug, was also tested for the purpose of comparison. After the behavioral procedures had been undertaken, the animals were killed and brain tissue was sampled to evaluate DNA damage in the brain using comet assay. The data reported here suggest that the administration of phenobarbital (10 mg/kg) and gamma-decanolactone at 0.3 g/kg, but not at 0.1 g/kg, impairs both the severity and the progression of seizures in the PTZ-kindling model. DNA damage to brain tissue decreased in gamma-decanolactone-treated kindling animals (similar to phenobarbital) as compared to nontreated animals. The results suggest that gamma-decanolactone has dose-dependent anticonvulsant properties, and may also have antiepileptogenic and neuroprotective effects in the PTZ-kindling model.

[Caffeine and antiepileptic drugs: experimental and clinical data]. / Kofeina a leki przeciwpadaczkowe: dane doswiadczalne i kliniczne.

BACKGROUND: Caffeine, a methylxanthine derivative, is contained in coffee or tea, chocolate as well as in some beverages. In addition, it may be added to some analgesics. At high doses, similarly to other methylxanthine derivatives (theophylline, pentoxifylline) caffeine induces seizure activity in rodents. THE AIM OF STUDY: If caffeine intake from coffee drinking resulting in pharmacologically active plasma caffeine concentrations--can lead to diverse interactions with other medications. RESULTS: Since 90s of the XX century, there are experimental data available pointing to the caffeine-induced impairment of the protective activity of a number of antiepileptic drugs in basic models of epilepsy in rodents. Acute caffeine, in doses far below its convulsive potential (almost 10-20 fold lower than the ED50 of the methylxanthine of 2.03 mmol/kg for the induction of seizures), produced a significant reduction in the anticonvulsant effects of carbamazepine, phenobarbital, phenytoin, and valproate against maximal electroshock-induced seizures in mice. This interaction was pharmacodynamic in nature since caffeine did not affect the plasma concentrations of these anti-epileptics. Interestingly, there was no tolerance to this hazardous effect of caffeine since its administration at the same dosages (0.12-0.24 mmollkg) also resulted in the impairment of the protection provided by antiepileptic drugs, this effect being even more pronounced in the case of phenobarbital and carbamazepine. In case of newer antiepileptics, both acute and chronic caffeine decreased the protective potential of gabapentin and topiramate but not that of lamotrigine and tiagabine. CONCLUSIONS: The existing clinical data confirm the experimental results in that caffeine intake in epileptic patients results in increased seizure frequency. It may be concluded that epileptic patients should limit their daily intake of caffeine.

Antioxidant and hepatoprotective effects of Eucalyptus maculata.

BACKGROUND: Since phenolic compounds have been reported as effective antioxidants, this study was designed to assess the hepatoprotective and antioxidant activities of the chloroformic extract of the resinous exudate and its phenolic constituents obtained from the stems of Eucalyptus maculata. MATERIAL/METHODS: The chloroformic extract and pure phenolic isolates were evaluated for their antioxidant and hepatoprotective properties in mice and rats based on biochemical changes in serum and tissues as well as pathological changes in the liver and spleen. RESULTS: Acetaminophen (ACP) at a dose of 1 g/kg body weight produced 100% mortality in mice, while pretreatment of animals with the chloroformic extract (125 and 250 mg/kg) protected against the moralities by 66%. Pretreatment of rats with either the chloroformic extract (250 mg/kg) or any of the pure isolates (20 mg/kg) significantly reduced the increase in serum level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) produced by ACP (640 mg/kg). Pretreatment of animals with the chloroformic extract or its isolates also protected against ascorbic acid depletion in serum and kidney tissues induced by oral administration of paraquat (PQ) without modifying the serum level of glutathione (GSH) and glycogen content in liver tissue. CONCLUSIONS: The phenolic content of the chloroformic extract and the pure isolates produced an antioxidant activity which may be due to the formation of stable phenoxyl radical in addition to its effect through vitamin C.

Nicotine therapy in adulthood reverses the synaptic and behavioral deficits elicited by prenatal exposure to phenobarbital.

A major objective in identifying the mechanisms underlying neurobehavioral teratogenicity is the possibility of designing therapies that reverse or offset drug- or toxicant-induced neural damage. In our previous studies, we identified deficits in hippocampal muscarinic cholinergic receptor-induced membrane translocation of protein kinase C (PKC)gamma as the likely mechanism responsible for adverse behavioral effects of prenatal phenobarbital exposure. We therefore explored whether behavioral and synaptic defects could be reversed in adulthood by nicotine administration. Pregnant mice were given milled food containing phenobarbital to achieve a daily dose of 0.5-0.6 g/kg from gestational days 9-18. In adulthood, offspring showed deficits in the Morris maze, a behavior dependent on the integrity of septohippocampal cholinergic synaptic function, along with the loss of the PKCgamma response. Phenobarbital-exposed and control mice then received nicotine (10 mg/kg/day) for 14 days via osmotic minipumps. Nicotine reversed the behavioral deficits and restored the normal response of hippocampal PKCgamma to cholinergic receptor stimulation. The effects were regionally specific, as PKCgamma in the cerebellum was unaffected by either phenobarbital or nicotine; furthermore, in the hippocampus, PKC isoforms unrelated to the behavioral deficits showed no changes. Nicotine administration thus offers a potential therapy for reversing neurobehavioral deficits originating in septohippocampal cholinergic defects elicited by prenatal drug or toxicant exposures.

Pretreatment with Ginkgo biloba extract weakens the hypnosis action of phenobarbital and its plasma concentration in rats.

In a previous study, we found that orally administered Ginkgo biloba extract (GBE) induced hepatic cytochrome P450 (CYP) in rats, especially the CYP2B type. This fact suggested that GBE influenced the availability and safety of drugs that were metabolized via CYP2B type enzymes. To confirm this possibility, in this study we examined the effect of feeding a 0.1, 0.5 and 1.0% GBE diet for 2 weeks on the pharmacokinetics and pharmacological action of phenobarbital, which is known to be metabolized by CYP2B in Wistar rats. The feeding of GBE markedly shortened the sleeping time in rats. Furthermore, the maximal phenobarbital plasma concentration (Cmax) and the 24-h area under the curve (AUC0-24) were decreased in rats fed GBE. These findings indicate that GBE reduces the therapeutic potency of phenobarbital via enhancement of cytochrome P450 expression, and raises the possibility that GBE and drug interactions may occur clinically.

Clinical pharmacokinetics of thalidomide

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions in being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (Cmax) of 1-2mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUCoo) of 18mg - h/L, apparent elimination half-life of 6 hours and apparent systemic clearence of 10 L/H. Thalidomide pharmacokinetics are best described by a one-comportment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacolinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than in absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state Cmax (Cssmax) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accululation, with Css of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400mg. Because of the low solubility of thalidomide Cmax is less than proportional to dose, and tmax is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokonetics are not expected to change in patients with impaired liver...

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

Nicotine diminishes anticonvulsant activity of antiepileptic drugs in mice.

Nicotine administered acutely at subconvulsive dose of 4 mg/kg, significantly decreased the protective activity of valproate, carbamazepine, diphenylhydantoin, phenobarbital, topiramate and lamotrigine against maximal electroshock-induced tonic convulsions in mice. The obtained data may suggest that interaction between nicotine and antiepileptic drugs should be carefully considered as a cause of the therapeutic failure in epileptic patients.

Niguldipine impairs the protective activity of carbamazepine and phenobarbital in amygdala-kindled seizures in rats.

There is evidence that some calcium (Ca(2+)) channel inhibitors enhance the protective activity of antiepileptic drugs. Since clinical trials have not provided consistent data on this issue, the objective of this study was to evaluate the interaction of a dihydropyridine, niguldipine, with conventional antiepileptics in amygdala-kindled rats. Niguldipine (at 7.5 but not at 5 mg/kg) displayed a significant anticonvulsant effect, as regards seizure and afterdischarge durations in amygdala-kindled convulsions in rats, a model of complex partial seizures. No protective effect was observed when niguldipine (5 mg/kg) was combined with antiepileptics at subeffective doses, i.e. valproate (75 mg/kg), diphenylhydantoin (40 mg/kg), or clonazepam (0.003 mg/kg). Unexpectedly, the combined treatment of niguldipine (5 mg/kg) with carbamazepine (20 mg/kg) or phenobarbital (20 mg/kg) resulted in a proconvulsive action. BAY k-8644 (an L-type Ca(2+) channel activator) did not modify the protective activity of niguldipine (7.5 mg/kg) or the opposite action of this dihydropyridine (5 mg/kg) in combinations with carbamazepine or phenobarbital. A pharmacokinetic interaction is not probable since niguldipine did not affect the free plasma levels of the antiepileptics. These data indicate that the opposite actions of niguldipine alone or combined with carbamazepine (or phenobarbital) were not associated with Ca(2+) channel blockade. The present results may argue against the use of niguldipine as an adjuvant antiepileptic or for cardiovascular reasons in patients with complex partial seizures.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5'-proximal region of rat liver CYP2B1/B2 gene into the nucleus.

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1/B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M(r) of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation.

Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture. Involvement of calcium- and cAMP-dependent pathways.

Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.

Transcriptional regulation of 5-aminolevulinate synthase by phenobarbital and cAMP-dependent protein kinase.

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.

Phosphorylation/Dephosphorylation steps are crucial for the induction of CYP2B1 and CYP2B2 gene expression by phenobarbital.

The effects of several protein kinase activators and protein phosphatase inhibitors on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes were investigated. Insulin, epidermal growth factor, interleukin 6, cAMP, phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, vanadate, and okadaic acid were found to suppress the induction of CYP2B1/2B2 at mRNA and protein levels in hepatocytes. cAMP and vanadate completely suppressed the induction of CYP2B1/2B2 gene expression in both rat hepatocytes and liver. The addition of genistein to vanadate-treated hepatocytes partially recovered the induction of CYP2B1/2B1 gene expression by PB. These results of the present study demonstrate that phosphorylation/dephosphorylation steps are crucial for the induction of CYP2B1/2B2 gene expression by PB.

Phenobarbital-mediated induction of the CYP2B subfamily is not antagonized by picrotoxin, a potent antagonist of barbiturates in the central nervous system.

Our previous study indicated that picrotoxin competes with phenobarbital (PB) for the binding to liver microsomes, although the target of binding and the physiological role were not determined (unpublished observation). To seek information on the target site of PB, reflecting the induction of hepatic enzymes, we examined here the effect of picrotoxin on PB-mediated induction of hepatic cytochrome P450 and UDP-glucuronosyltransferase in vivo in rats. The induction of the CYP2B1/2 was estimated by immunoblot analysis and by measuring the activity of testosterone 16beta-hydroxylation. Intraperitoneal injection of picrotoxin alone slightly increased CYP2B1/2 protein. An experiment on co-treatment of picrotoxin and PB showed that picrotoxin enhanced rather than antagonized the inducing effect of PB. The results suggest two possibilities: that 1) picrotoxin increases the CYP2B subfamily by binding to the same site as PB; or 2) the site in microsomes for the competition between PB and picrotoxin does not reflect the induction of P450.

Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes.

Our laboratory has proposed that phenobarbital (PB), a typical lipophilic agent that induces some members of the supergene family of liver microsomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disrupted cytoskeleton. To verify that these manipulations of the culture environment block specific steps in the PB induction pathway rather than simply exerting nonspecific or toxic effects on CYP2B1/2 gene transcription, we have now examined PB induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a "nonclassical" pathway apparently involving the glucocorticoid receptor. We found that in primary cultures of adult rat hepatocytes treated with PB, induction of CYP3A23 mRNA, just as we reported for induction of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted basement membrane) and was blocked by the presence of cytoskeletal inhibitors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent protein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditions that block its induction by PB. We conclude that the effects of extracellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, pretranscriptional steps in the pathway(s) for PB induction of liver cytochromes P450.

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.

Transgenic mouse models in carcinogenesis: interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis.

1. Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumours, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. 2. Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human transforming growth factor (TGF-alpha) cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumourigenesis. 3. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumours were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. 4. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. 5. We have now extended these studied and examined the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumour promotion by phenobarbitone was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbitone was an effective tumour promoter in the c-myc single transgenic mice. 6. The results indicate that HGF may function as a tumour suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.

Effect of alpha-hederin and sapindoside B on hepatic microsomal cytochrome P-450 in mice.

AIM: To study the relation between the effect of alpha-hederin (Hed) and sapindoside B (Sap B) on cytochrome P-450 and their hepatoprotection. METHODS: Mice were given sc Hed, Sap B, as well as a mixture of Hed + Sap B (1:1.5), and hepatic microsomal cytochrome P-450 was examined. RESULTS: Hed 20 mg.kg-1, Sap B 20 mg.kg-1 and Hed + Sap B 20 mg.kg-1 sc, reduced hepatic P-450 content by 40%, 55% and 50%, respectively. The effect of the saponins on P-450 was reversible, as P-450 level returned to normal after 3 d. Phenobarbital (Phe) 50 mg.kg-1 ip increased P-450 2.5-fold, the Phe-induced P-450 was inhibited 50% by Hed + Sap B. No inhibitory effect was seen when liver microsomes were incubated with Hed + Sap B in vitro. CONCLUSION: The hepatoprotective effects of Hed and Sap B were at least in part, due to its suppressive effect on liver cytochrome P-450.

Effects of phenobarbital and interleukin-6 on cytochrome P4502B1 and 2B2 in cultured rat hepatocytes.

The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL-6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process.