Molecular characterization of tomato 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase.

Analysis of cDNAs encoding the bifunctional 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase (DHQase-SORase) from tomato (Lycopersicon esculentum) revealed two classes of cDNAs that differed by 57 bp within the coding regions, but were otherwise identical. Comparison of these cDNA sequences with the sequence of the corresponding single gene unequivocally proved that the primary transcript is differentially spliced, potentially giving rise to two polypeptides that differ by 19 amino acids. Quantitative real-time polymerase chain reaction revealed that the longer transcript constitutes at most 1% to 2% of DHQase-SORase transcripts. Expression of the respective polypeptides in Escherichia coli mutants lacking the DHQase or the SORase activity gave functional complementation only in case of the shorter polypeptide, indicating that skipping of a potential exon is a prerequisite for the production of an enzymatically active protein. The deduced amino acid sequence revealed that the DHQase-SORase is most likely synthesized as a precursor with a very short (13-amino acid) plastid-specific transit peptide. Like other genes encoding enzymes of the prechorismate pathway in tomato, this gene is elicitor-inducible. Tissue-specific expression resembles the patterns obtained for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 2 and dehydroquinate synthase genes. This work completes our studies of the prechorismate pathway in that cDNAs for all seven enzymes (including isozymes) of the prechorismate pathway from tomato have now been characterized.

Public information and the ethical responsibility of the industry.

Biotechnology and gene technology are recognized by experts as invaluable and unique tools to find solutions to or improve many problems in health, agriculture and management of the environment, and are regarded as a driving economic force in the next century. They are, however, by large not accepted by the public and the discussion on gene technology is often emotional or even controversial. This situation is not favorable to a constructive and open debate. Science, economic forces and society coexist symbiotically and a broad debate on all aspects including the social and ethical issues of biotechnology is essential. A mutual understanding and acceptance are prerequisites to the democratic process of the elaboration of sound regulation and appropriate administration. To this aim, a task force was initiated in Switzerland by the pharmaceutical industry. Its goal is to provide information on relevant aspects of biotechnology and to participate in the dialogue with the Swiss public and politicians on critical issues like risk assessment, ethics, safety, novel food and legislation. A documentation service, teaching material, national poster campaign, exhibitions and debates on specific issues are the contributions of the industry to a fair information in favor of the improved background to an educated and open debate.

Peroxisome proliferators and cellular signaling pathways. A review.

In a variety of biological systems, the cellular response to an extracellular stimulus is mediated by a complex cascade of biochemical signals transduced from the cellular membrane to the specific part(s) of the cell where the response(s) occur(s). The signal transduction pathways do form a matrix of several reactions involving increased intracellular free Ca2+ levels, activation of Na+/H+ exchange, stimulation of phosphatidylinositol turnover, activation of protein kinase C and increased transcription of several cellular proto-oncogenes. Each of them is subject to modulation at many levels, and is also susceptible to deregulation during chemically-induced carcinogenesis. It is the aim of this short review to give an insight into the complexity of this system which may mediate the effects induced in hepatocytes by peroxisome proliferators or may, upon chronic exposure, be altered by some of them.

Toxicity of peroxisome proliferators.

Peroxisome proliferators are not a chemical class of compounds. They do not have a similar chemical structure but all induce characteristic effects in the liver of treated rats or mice. They produce within a few days a striking dose-dependent hepatomegaly accompanied by a characteristic proliferation of the peroxisomal and microsomal compartment as assessed morphologically and biochemically. Such effects are not observed in other species including human. In addition, life-long feeding of the susceptible laboratory animals results in the formation of liver tumor. The effects induced in subchronic studies can be reproduced and investigated in cultured hepatocytes, the target cells. The species specificity is observed with all peroxisome proliferators, and by large the effects observed in subchronic studies are reversible. The hepatocarcinogenesis by peroxisome proliferators is not fully understood, because these compounds are not directly genotoxic, but the understanding of their tumor promotor potential has some implications for the toxicological testing and risk assessment.

Effect of propaquizafop and its free-acid derivative on lauric acid hydroxylation and peroxisomal beta-oxidation in primary cultured rat, mouse, guinea pig and marmoset hepatocytes.

The effects of the phenoxyisopropionic acid derivative propaquizafop and its free-acid derivative on microsomal lauric acid hydroxylase and peroxisomal fatty acyl-CoA beta-oxidation have been investigated in primary cultured hepatocytes obtained from various species and compared with those induced by bezafibrate. The hepatocyte cultures were incubated with these compounds for 48 hr at concentrations between 0.1-100mum. No signs of cytotoxicity were observed as shown by the lack of lactate dehydrogenase (LDH) release into the culture medium. Lauric acid 12-hydroxylase was found to be strongly induced after treatment of rat and mouse hepatocytes with all three compounds, but remained largely unaffected in guinea pig and marmoset hepatocytes. Concomitantly, peroxisomal fatty acyl-CoA beta-oxidation was found to be induced in rat but not in mouse hepatocytes after treatment for 48 hr. However, clearly increased beta-oxidation activities could also be observed in mouse hepatocytes after a 72-hr incubation period. In contrast, only marginally increased beta-oxidation activities were recorded, if at all, in guinea pig and marmoset hepatocytes. The results demonstrate that the effects of propaquizafop and its free-acid derivative in hepatocytes from four species are very similar to those produced by the known peroxisome proliferator (PP) bezafibrate. This is in accordance with the known difference in susceptibility of various species to PP, for example, rats and mice being most responsive whereas guinea pigs and primates including humans are far less responsive or even unresponsive.

Studies on the mechanism of induction of microsomal cytochrome P452 and peroxisomal bifunctional enzyme mRNAs by nafenopin in primary cultures of adult rat hepatocytes.

The amount of the two mRNAs although lower in cultured hepatocytes than in freshly isolated cells was found to be rapidly inducible upon the addition of 32 microM nafenopin. The induction of cyt.P452 mRNA always preceded the induction of PBE mRNA, but for both, the maximal induction (10-20-fold over control) was obtained within 24 hr and was achieved by transcriptional activation. At early time points (1 and 2 hr after the addition of nafenopin), in the absence of on-going protein synthesis, the amount of cyt.P452 mRNA (and not of PBE mRNA) was transiently higher in the presence of cycloheximide and nafenopin than in the presence of nafenopin alone.

Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study.

Ultrastructural changes were investigated and quantified, using a stereological approach, in early gamma-glutamyltranspeptidase (GGT)-positive focal lesions, induced in the rat liver by treatment with a single initiating dose of diethylnitrosamine (DENA) followed by promotion with phenobarbitone (PB) for 30 weeks. Within the extra-hepatocyte environment of focal tissue, the mean volume occupied by Ito cells was markedly decreased, whilst that occupied by endothelial and Kupffer cells was increased, when compared to uninvolved tissue from the same rat livers. The bile canaliculi were dilated, but no significant differences in the mean volume occupied by the sinusoidal and Disse spaces were noted. In focal hepatocytes there was a striking overproduction of lipid droplets and proliferation of smooth endoplasmic reticulum (sER). Whorls of concentrically arranged, parallel ER membranes were found only in the hepatocytes of preneoplastic foci, in association with the proliferated sER, and never in the surrounding, uninvolved tissue. The increase in mean volume of the sER, lipid droplet and cytoplasmic matrix compartments, together with the appearance of whorls, were the major contributing factors to the marked hypertrophy seen in focal hepatocytes. The mean volume of the rough endoplasmic reticulum, mitochondrial, lysosomal, peroxisomal and nuclear compartments per hepatocyte also increased, but contributed to a lesser extent to the cellular hypertrophy. It is speculated that whorls may be structural adaptations, resulting from a possible alteration in the normal feedback control of cholesterol synthesis, for the production of sterols and the biogenesis of sER in eosinophilic-type focal cells. The significance of changes observed in focal tissue, and the high biological variation noted between foci, is discussed in relation to the hepatocarcinogenic process.

Increase in hepatocyte and nuclear volume and decrease in the population of binucleated cells in preneoplastic foci of rat liver: a stereological study using the nucleator method.

Gamma-glutamyltranspeptidase-positive hepatocyte foci were produced in female rats given a single dose of diethylnitrosamine neonatally after birth and, after weaning, a diet containing phenobarbitone for 30 wk. The nucleator method, a new stereological approach, provided an efficient, unbiased estimate of mean cell volume in focal lesions and extrafocal areas. It also provided an unbiased sample of cells to estimate hepatocyte nuclear volume and the percentage of binucleated cells. The results showed an increase in the mean volume of mononucleated cells--from 4,700 micron3 in extrafocal areas to 12,700 micron2 in foci--and of binucleated cells--from 6,900 micron3 to 25,000 micron3. This demonstrated the hypertrophic effect of the carcinogenic treatment in focal lesions. A striking reduction in the proportion of binucleated cells was also observed in the preneoplastic lesions. Nuclear volume measurements from mononucleated and binucleated hepatocytes were used to assess ploidy. An apparent increase in nuclear ploidy, with no change in cellular ploidy, was noted in focal tissue when compared with nonfocal tissue. This appeared to be caused by an increase in mononucleated tetraploid cells and a reduction in binucleated cells with two diploid nuclei, indicating an altered mitotic mechanism in focal lesions. The significance of these changes in cell volume, apparent ploidy levels and binuclearity in preneoplastic foci is discussed in relation to the hepatocarcinogenic process.

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro.

[14C]Di-n-octyltin dichloride ([14C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = mumol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNA-binding ability is about 30,000 times lower than the corresponding value for the strong carcinogen, aflatoxin B1. In addition, [14C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.

[Use of hepatocytes in primary cultures to predict potential hepatocarcinogenicity of compounds]

Short-term tests for genotoxic activity will detect a variety of potential carcinogens. However, experience over the last few years has shown that many chemical carcinogens are not detected in these tests. A large proportion of these non-mutagenic (non-genotoxic) carcinogens induce tumors in the rodent liver after life-long feeding. One class of such carcinogens are the hypolipidemic drugs many of which induce a characteristic hepatomegaly upon subchronic treatment. Typically this liver enlargement is accompanied by increased mitotic activity and a proliferation of the hepatic peroxisome compartment. Results also suggest a correlation between the degree and the nature of the hepatomegalic response to a compound, and its hepatocarcinogenic potency, but it remains unclear whether the growth stimulation and/or the peroxisome proliferation is correlated with the carcinogenicity. Both, the induction of peroxisomal enzyme and of replicative DMA synthesis may be measured in primary cultures of adult rat hepatocytes following in vitro exposition. Moreover, the ability of the compounds tested to induce in vitro replicative DNA synthesis in primary cultures of adult rat hepatocytes correlated well with the potency of the hepatomegalic response induced in vivo in treated rodents. Consequently, studies using such cultures might be useful to characterize and possibly predict in vitro the hepatocarcinogenic potency of some new compounds. The early recognition of the possible carcinogenic property of a new substance might accordingly contribute to the reduction of a number of life-long studies.

The significance of in vitro studies on peroxisome proliferation.

Peroxisome proliferation in rodents is associated with hepatocarcinogenicity. This association has led to increased interest in the phenomenon and to the search for in vitro tests to detect peroxisome proliferators and to study the mechanisms by which proliferation occurs. Primary cultures of adult rat hepatocytes provide such a system: peroxisomal enzymes, cytochrome P-452 and replicative DNA synthesis may all be induced and the response to treatment with many peroxisome proliferators is observed in a manner similar to that in the liver in vivo. Cultured hepatocytes, therefore, provide an optimal system: (i) to screen for potential peroxisome proliferators; (ii) to study structure-activity relationships; (iii) to investigate species differences in the effects of peroxisome proliferators on hepatocytes; and (iv) to study the molecular mechanisms underlying peroxisome proliferation and its relationship to tumour formation.

Potentiation of diacylglycerol-activated protein kinase C by acyl-coenzyme A thioesters of hypolipidaemic drugs.

Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, bezafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca+2, phosphatidylserine and diolein, thus differentiating their action from that of other tumor promoters such as phorbol esters. Under similar conditions the unesterified drugs were comparatively ineffective. Similar results were obtained using the rat liver enzyme. The data suggest that acylcoenzyme A thioesters of hypolipidaemic drugs, may play a role in the induction of liver tumors by these compounds, through the potentiation of protein kinase C.

Hydrolysis of bisphenol A diglycidylether by epoxide hydrolases in cytosolic and microsomal fractions of mouse liver and skin: inhibition by bis epoxycyclopentylether and the effects upon the covalent binding to mouse skin DNA.

Synergistic interactions have been reported in the carcinogenicity of two epoxy resin components to mouse skin. A mixture of bisphenol A diglycidylether and bis epoxycyclopentylether was highly carcinogenic, despite the fact that neither compound gave positive results when applied individually. To elucidate the mechanism of this synergistic interaction we have investigated the effects of bis epoxycyclopentylether upon the hydrolysis and DNA-binding of bisphenol A diglycidylether. This glycidylether was rapidly hydrolysed by microsomal and cytosolic fractions of mouse liver and skin. In three different mouse strains the specific epoxide hydrolase activities were 28.3-48.5; 33.0-38.8; 7.9-10.2 and 0.85-0.98 nmol/mg protein/min for liver microsomal and cytosolic and skin microsomal and cytosolic fractions respectively. This is the first demonstration of an epoxide hydrolase activity in skin cytosolic fractions. Bis epoxycyclopentylether inhibited the microsomal activities. This inhibition appeared to be slightly more effective with microsomal fractions from liver. The effect of this inhibition upon the binding of bisphenol A diglycidylether to mouse skin DNA was investigated using bisphenol A diglycidylether radiolabelled at two different positions. When high doses of bisphenol A diglycidylether were applied to the mouse skin one major DNA adduct was observed which was identified as a glycidaldehyde adduct. This adduct was not detectable at the lowest bisphenol A diglycidylether dose tested, unless bis epoxycyclopentylether was applied simultaneously. These findings suggest that glycidaldehyde may be formed from bisphenol A diglycidylether. At low doses, however, the epoxide groups are hydrolysed before glycidaldehyde can be formed, unless the epoxide hydrolase is inhibited. Such inhibition and the associated increased production of glycidaldehyde may account for the potentiation of the carcinogenic response in the epoxide mixture.

Stimulation of DNA synthesis but not of peroxisomal beta-oxidation by nafenopin in primary cultures of marmoset hepatocytes.

The effects of the peroxisome proliferator nafenopin upon primary cultures of marmoset hepatocytes have been investigated and compared to those on cultured rat hepatocytes. Nafenopin did not induce peroxisomal beta-oxidation or peroxisome proliferation but did induce replicative DNA synthesis. These findings demonstrate that peroxisome proliferation and mitogenicity are two independent properties of nafenopin and question the widely held view that primates are generally insensitive to the effects of peroxisome proliferators.

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor.

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation.

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice.

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.

Increased peroxisomal enzyme mRNA levels in adult rat hepatocytes cultured in a chemically defined medium and treated with nafenopin.

Nafenopin is a known inducer of peroxisome proliferation in the hepatocytes of treated rodents. Primary cultures of adult rat hepatocytes maintained in a chemically-defined medium respond to the drug. RNAs from hepatocyte cultures treated for 1, 8 and 20 hr and their untreated counterparts have been purified and hybridized to radioactive cDNA probes specific for peroxisomal mRNAs (for catalase and the three enzymes of the ß-oxidation system). The amount of the specific mRNAs was fairly constant or increased slightly in control cultures, but increased steadily during treatment of the cultures with a non-toxic dose of nafenopin (32 µm). For the peroxisomal bifunctional enzyme mRNA, representative of the ß-oxidation system, this increase was approximately fivefold after 20 hr, whereas for catalase mRNA a twofold increase compared with the control was observed after 20 hr. The time-course of the induction of the peroxisomal bifunctional enzyme mRNA in vitro was found to be similar to that observed after intragastric treatment of rats with nafenopin. This indicates that mechanistic studies on the early events induced in hepatocytes by peroxisome proliferators can be performed with this culture system. Such studies may help to explain the hepatotoxic/hepatocarcinogenic properties of this class of xenobiotics.

Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: further characterization and effect of nafenopin, a peroxisome proliferator.

The addition of 2% dimethyl sulfoxide to adult rat hepatocytes cultured in a chemically defined medium at Day 1 after cell plating resulted in maintenance of the cytochrome P-450 content and the cyanide-insensitive palmitoyl-CoA beta-oxidation activity at 66 and 70% of the initial Day 1 values. The addition of phenobarbital, 3-methylcholanthrene, or nafenopin from Day 3 to Day 6 increased the contents of cytochrome P-450 to 128, 239, and 251%, respectively, compared to untreated controls at Day 3. In addition, nafenopin also caused a pronounced and time-dependent increase in palmitoyl-CoA beta-oxidation activity but was found to have only a weak stimulating effect on replicative DNA synthesis (2-fold) when compared to that of epidermal growth factor (6.5-fold). In the presence of dimethyl sulfoxide the hepatocyte cultures could be kept alive for more than 1 month. Exposure of such cultures to nafenopin from Day 1 do Day 37 resulted in survival which was even better than that of their untreated counterparts. This effect was accompanied by the appearance of abundant endoplasmic reticulum membranes and an increased number of peroxisomes.

Assessment of peroxisome proliferation and liver growth-stimulating potential by nondirectly genotoxic compounds in cultured hepatocytes.

Previously, we have established that some peroxisome proliferators, a class of nongenotoxic hepatocarcinogens, are able to induce replicative DNA synthesis (RDS) in cultured hepatocytes. Hepatomegaly observed after short-term in vivo treatment correlated better with the ability to induce RDS than with the potency as peroxisome proliferator assessed in vitro. To clarify the challenging question of the limited sensitivity of primates to peroxisome proliferators, primary cultures of marmoset hepatocytes have been treated with nafenopin for some days. As expected from in vivo observations, no evidence for peroxisome proliferation could be observed. However, nafenopin induced a dose-dependent increase in the amount of RDS, but this induction was measurable only when the serum was absent from the culture medium. These results confirm that peroxisome proliferation and mitogenicity might be independent properties of peroxisome proliferators. Since in vivo the ability of compounds to induce RDS in liver cells is relevant to at least one key parameter of the hepatocarcinogenic response, it is suggested that measurement of RDS inducibility in cultured hepatocytes from different species might be relevant and useful to assess species differences in the liver tumor potency of nondirectly genotoxic compounds.