PPARalpha: mechanism of species differences and hepatocarcinogenesis of peroxisome proliferators

Peroxisome proliferator chemicals are classic non-genotoxic carcinogens. These agents cause liver cancers when chronically administered to rats and mice. Peroxisome proliferators include the widely prescribed lipid and cholesterol lowering fibrate drugs. In contrast to the results in rodents, there is no evidence that fibrates are associated with elevated risk of liver cancer or any other neoplasms in humans thus indicating a species difference in the hepatocarcinogenic response. The biological effects of peroxisome proliferators are mediated by the peroxisome proliferator-activated receptor (PPAR)alpha. Pparalpha-null mice are resistant to all of the pleiotropic effects of peroxisome proliferators, including cell proliferation and hepatocarcinogenesis. The mechanism of hepatocellular proliferation involves downregulation of the microRNA let-7c gene by PPARalpha. Let-7c controls levels of proliferative c-myc by destabilizing its mRNA. Thus, upon suppression of let-7c, c-myc mRNA and protein are elevated resulting in enhanced hepatocellular proliferation. In contrast, PPARalpha-humanized mice, that respond to Wy-14,643 by lower serum triglycerides and induction of genes encoding fatty acid metabolizing enzymes, are resistant to peroxisome proliferator-induced cell proliferation and cancer. These mice do not exhibit downregulation of let-7c gene expression thus forming the basis for the resistance to hepatocellular carcinogenesis.     

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor β1 (TGFβ1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal β-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of β-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents. Received: 16 August 1999 / Accepted: 21 September 1999     

Investigation of species differences in isobutene (2-methylpropene) metabolism between mice and rats

Metabolism of isobutene (2-methylpropene) in rats (Sprague Dawley) and mice (B6C3F₁) follows kinetics according to Michaelis-Menten. The maximal metabolic elimination rates are 340 mol/kg/h for rats and 560 mol/kg/h for mice. The atmospheric concentration at which V_max/2 is reached is 1200 ppm for rats and 1800 ppm for mice. At steady state, below atmospheric concentrations of about 500 ppm the rate of metabolism of isobutene is direct proportional to its concentration. 1,1-Dimethyloxirane is formed as a primary reactive intermediate during metabolism of isobutene in rats and can be detected in the exhaled air of the animals. Under conditions of saturation of isobutene metabolism the concentration of 1,1-dimethyloxirane in the atmosphere of a closed exposure system is only about 1/15 of that observed for ethene oxide and about 1/100 of that observed for 1,2-epoxy-3-butene as intermediates in the metabolism of ethene or 1,3-butadiene. On leave from: Central Research Institute of Chemistry, Hungarian Academy of Sciences, Budapest, Hungary.     

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB)

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor β1 (TGFβ1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFβ1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis. Received: 9 June 1998 / Accepted: 23 September 1998     

Sulfation of melatonin: Enzymatic characterization,differences of organs,species and genders,and bioactivity variation

Exogenous melatonin (Mel) is widely used in clinic for multiple therapeutic purposes. In metabolism pathways of Mel, 6-hydroxymelatonin-sulfate (S-O-Mel) and N-acetylserotonin sulfate (S-NAS) are the most abundant metabolites account for over 90% of total Mel metabolites in humans, indicating that sulfation plays an important role in reflecting the functions and clearance of Mel in vivo. In the present study, we characterized Mel sulfation using various human organ cytosols (liver, lung, kidney, small intestine and brain), liver cytosols from five different animal species, and cDNA-expressed human sulfotransferase (SULT) for the first time. Our results demonstrated that liver, lung, kidney and small intestine of humans had high catalytic efficiency for Mel sulfation, however, brain contained a very low reaction rate. Interestingly, organ cytosols prepared from females exhibited higher sulfation activity than those of males. SULT isoforms 1A1, 1A2, 1A3, 1B1 and 1E1 exhibited metabolic activities toward Mel. According to kinetic parameters (K_m and V_max), chemical inhibition, correlation analysis, molecular docking and sulfation assays with recombinant human SULTs isoforms, SULT1A1 was determined as the major enzyme responsible for Mel sulfation. Furthermore, considerable species differences in Mel sulfation were observed, and the total intrinsic clearance rate of Mel sulfation was as follows: monkey > rat > dog > human > pig > mouse. Additionally, the anti-inflammatory effects of Mel and its sulfated metabolites were evaluated by inhibiting nitric oxide (NO) production in RAW264.7 cells, and S-O-Mel as a bioactive form, exhibited potent bioactivity. Our investigation provided a global view of the enzyme-dependent sulfation of Mel that can guide biomedical research on Mel.     

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial β-oxidation and the activity of catalase,glutathione S-transferase and glutathione peroxidase

The effect of hypolipidaemic compounds on peroxisomal fatty acid β-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial β-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial β-oxidation activities in intact cells. Overall β-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate β-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal β-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate β-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells. When a comparison between both species is made for the ratio of hydrogen peroxide production over catalase activity, it can be concluded that this peroxide will have much smaller possibilities to escape from the peroxisomal compartment in monkey hepatocytes. These findings suggest that species differences in these enzyme activities can contribute to differences in susceptibility for peroxisome proliferator-induced carcinogenicity between rodents and primates. Received: 3 January 1994/Accepted: 11 April 1994     

Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors

The kinetic and pharmacological characteristics of the binding of [3H]ketanserin and [3H]mesulergine to frontal cortical brain membranes from rat, pig and human were studied. In the 3 species [3H]ketanserin labeled sites with the characteristics of the 5-HT2 receptors previously described in the rat. In contrast, [3H]mesulergine labeled 5-HT2 receptors in rat, but not in pig and human cortices. The characteristics of the sites labeled by [3H]mesulergine in pig cortex were similar to those of sites in the choroid plexus of rats, pigs and humans. While several reputed 5-HT2 ligands presented a similar affinity for the [3H]ketanserin binding sites in the 3 species, other such ligands, e.g. mesulergine, methysergide, cinanserin and LSD which displaced these sites with high affinity in rat brain, had lower affinities in pig and human brain. These results indicate that 5-HT2 receptors show different pharmacological profiles in different species. Caution should thus be exerted in extrapolating data from laboratory animals to humans.     

The effects of di 2-ethyl hexyl phthalate (DEHP) on cellular lipid accumulation in HepG2 cells and its potential mechanisms in the molecular level

Diethylhexyl phthalate (DEHP) is suspected to be an inevitable factor related to metabolic disease. Our previous study demonstrated that excess DEHP could exacerbate non-alcoholic fatty liver disease (NAFLD) in SD rats. Addressing the terra incognita in DEHP-induced metabolic dysfunction, this study used HepG2 cells to investigate the potential mechanisms involved in DEHP-induced toxicity in vitro. The cells were established lipid overload model with oleic acid and BSA, then exposed to different concentrations (5, 10, 25, 50, 100?μmol/l DEHP) of DEHP for further analysis. The Oil Red O staining results showed that DEHP could promote lipid accumulation in cells. The level of superoxide dismutase (SOD) and malondialdehyde (MDA) changed suggested the balance of oxidative stress was disrupted. Additionally, western blot analysis showed that DEHP could promote the expression of peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein 1c (SREBP-1c). By quantifying the expressions of the two proteins, it is of interest to determine that DEHP could promote lipid accumulation in hepatocytes via activating the SREBP-1c and PPARα-signaling pathway.     

Species differences in the hepatic effects of inducers of CYP2B and CYP4A subfamily forms: relationship to rodent liver tumour formation

1. Phenobarbitone and related compounds induce hepatic microsomal cytochrome P450 (CYP) 2B forms (mediated by the constitutive androstane receptor), whereas peroxisome proliferators induce CYP4A forms (mediated by the peroxisome proliferator-activated receptor alpha) in rats and mice.

2. A number of non-genotoxic CYP2B and CYP4A inducers have been shown to produce liver tumours in rats and mice.

3. The hepatic effects of CYP2B and CYP4A inducers are reviewed and evaluated with respect to their established modes of action for rodent liver tumour formation and species differences in response. While CYP2B and CYP4A inducers stimulate replicative DNA synthesis in rodent liver, they do not appear to be mitogenic agents in human hepatocytes.

4. Epidemiological studies have demonstrated that phenobarbitone and rodent peroxisome proliferators do not increase the incidence of liver tumours in humans.

5. It is concluded that rodent CYP2B and CYP4A inducers do not pose a hepatocarcinogenic hazard for humans.

     

Species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) in several organs of mice, rats, and marmosets

To clarify species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) we measured the activity of four DEHP-metabolizing enzymes (lipase, UDP-glucuronyltransferase (UGT), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH)) in several organs (the liver, lungs, kidneys, and small intestine) of mice (CD-1), rats (Sprague–Dawley), and marmosets (Callithrix jacchus). Lipase activity, measured by the rate of formation of mono(2-ethylhexyl) phthalate (MEHP) from DEHP, differed by 27- to 357-fold among species; the activity was highest in the small intestines of mice and lowest in the lungs of marmosets. This might be because of the significant differences between Vmax/Km values of lipase for DEHP among the species. UGT activity for MEHP in the liver microsomes was highest in mice, followed by rats and marmosets. These differences, however, were only marginal compared with those for lipase activity. ADH and ALDH activity also differed among species; the activity of the former in the livers of marmosets was 1.6–3.9 times greater than in those of rats or mice; the activity of the latter was higher in rats and marmosets (2–14 times) than in mice. These results were quite different from those for lipase or UGT activity. Because MEHP is considered to be the more potent ligand to peroxisome proliferator-activated receptor involved in different toxic processes, a possibly major difference in MEHP-formation capacity could be also considered on extrapolation from rodents to humans.     

Liquiritigenin decreases selective molecular and behavioral effects of cocaine in rodents

Cocaine, as an indirect dopamine agonist, induces selective behavioral and physiological events such as hyperlocomotion and dopamine release. These changes are considered as consequences of cocaine-induced molecular adaptation such as CREB and c-Fos. Recently, methanolic extracts from licorice was reported to decrease cocaine-induced dopamine release and c-Fos expression in the nucleus accumbens. In the present study, we investigated the effects of liquiritigenin (LQ), a main compound of licorice, on acute cocaine-induced behavioral and molecular changes in rats. LQ attenuated acute cocaine-induced hyperlocomotion in dose-dependent manner. In addition, LQ inhibited CREB phosphorylation and c-Fos expression in the striatum and the nucleus accumbens induced by acute cocaine. Results provide strong evidence that LQ effectively attenuates the acute behavioral effects of cocaine exposure and prevents the induction of selective neuroadaptive changes in dopaminergic signaling pathways. Further investigation of LQ from licorice extract might provide a novel therapeutic strategy for the treatment of cocaine addiction.     

"Behavioural despair" in rats and mice: strain differences and the effects of imipramine

Rats and mice when forced to swim in a restricted space will rapidly cease attempts to escape and become immobile. Previous experiments have shown that immobility was selectively reduced by antidepressant agents. The present experiments show that important differences exist between strains in both the amount of immobility observed and the effects of imipramine. Strain differences should therefore be taken into account in attempts to replicate results from one laboratory to another.