The metabolism, toxicity and results of tests for carcinogenicity have been reviewed with respect to the safety for humans of coumarin present in foodstuffs and from fragrance use in cosmetic ...products. Coumarin is a natural product which exhibits marked species differences in both metabolism and toxicity. The majority of tests for mutagenic and genotoxic potential suggest that coumarin is not a genotoxic agent. The target organs for toxicity and carcinogenicity in the rat and mouse are primarily the liver and lung. Moreover, the dose–response relationships for coumarin-induced toxicity and carcinogenicity are non-linear, with tumour formation only being observed at high doses which are associated with hepatic and pulmonary toxicity. Other species, including the Syrian hamster, are seemingly resistant to coumarin-induced toxicity. There are marked differences in coumarin metabolism between susceptible rodent species and other species including humans. It appears that the 7-hydroxylation pathway of coumarin metabolism, the major pathway in most human subjects but only a minor pathway in the rat and mouse, is a detoxification pathway. In contrast, the major route of coumarin metabolism in the rat and mouse is by a 3,4-epoxidation pathway resulting in the formation of toxic metabolites. The maximum daily human exposure to coumarin from dietary sources for a 60-kg consumer has been estimated to be 0.02
mg/kg/day. From fragrance use in cosmetic products, coumarin exposure has been estimated to be 0.04
mg/kg/day. The total daily human exposure from dietary sources together with fragrance use in cosmetic products is thus 0.06
mg/kg/day. No adverse effects of coumarin have been reported in susceptible species in response to doses which are more than 100 times the maximum human daily intake. The mechanism of coumarin-induced tumour formation in rodents is associated with metabolism-mediated, toxicity and it is concluded that exposure to coumarin from food and/or cosmetic products poses no health risk to humans.
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.
A number of non-genotoxic CYP2B and CYP4A inducers have been shown to produce liver tumours in rats and mice.
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.
Epidemiological studies have demonstrated that phenobarbitone and rodent peroxisome proliferators do not increase the incidence of liver tumours in humans.
It is concluded that rodent CYP2B and CYP4A inducers do not pose a hepatocarcinogenic hazard for humans.
This exploratory phase II study evaluated the safety and efficacy of belatacept in de novo adult liver transplant recipients. Patients were randomized (N = 260) to one of the following ...immunosuppressive regimens: (i) basiliximab + belatacept high dose HD + mycophenolate mofetil (MMF), (ii) belatacept HD + MMF, (iii) belatacept low dose LD + MMF, (iv) tacrolimus + MMF, or (v) tacrolimus alone. All received corticosteroids. Demographic characteristics were similar among groups. The proportion of patients who met the primary end point (composite of acute rejection, graft loss, death by month 6) was higher in the belatacept groups (42–48%) versus tacrolimus groups (15–38%), with the highest number of deaths and grafts losses in the belatacept LD group. By month 12, the proportion surviving with a functioning graft was higher with tacrolimus + MMF (93%) and lower with belatacept LD (67%) versus other groups (90%: basiliximab + belatacept HD; 83%: belatacept HD; 88%: tacrolimus). Mean calculated GFR was 15–34 mL/min higher in belatacept‐treated patients at 1 year. Two cases of posttransplant lymphoproliferative disease and one case of progressive multifocal leukoencephalopathy occurred in belatacept‐treated patients. Follow‐up beyond month 12 revealed an increase in death and graft loss in another belatacept group (belatacept HD), after which the study was terminated.
This study reports results of an exploratory phase II trial in adult liver transplant recipients to evaluate belatacept compared with tacrolimus‐based immunosuppression, concluding that a safe and effective belatacept regimen for use in liver transplant patients is not yet identified. See editorial by Knechtle and Adams on page 1717.
Abstract A large number of drugs and other chemicals have been shown to induce hepatic microsomal cytochrome P450 (CYP) forms in experimental animals and humans. Most CYP forms are induced by ...receptor-mediated mechanisms leading to an increase in gene transcription. Important nuclear receptors involved in the induction of CYP1A, CYP2B, CYP3A and CYP4A subfamily forms comprise, respectively, the aryl hydrocarbon receptor, the constitutive androstane receptor, the pregnane X receptor and the peroxisome proliferator-activated receptor alpha. Hepatic CYP form induction can be assessed by in vivo , ex vivo and in vitro methods. Significant species differences can exist in the enzyme induction response to a given chemical and also in the toxicological consequences of induction. Hepatic CYP form induction in humans may lead to clinically important drug–drug interactions. In rodents hepatic CYP form induction can be associated with the formation of tumours by non-genotoxic modes of action in the liver, thyroid and other tissues.
Light intensity and atmospheric CO2 partial pressure are two environmental signals known to regulate stomatal numbers. It has previously been shown that if a mature Arabidopsis leaf is supplied with ...either elevated CO2 (750 ppm instead of ambient at 370 ppm) or reduced light levels (50 μmol m−2 s−1 instead of 250 μmol m−2 s−1), the young, developing leaves that are not receiving the treatment grow with a stomatal density as if they were exposed to the treatment. But the signal(s) that it is believed is generated in the mature leaves and transmitted to developing leaves are largely unknown. Photosynthetic rates of treated, mature Arabidopsis leaves increased in elevated CO2 and decreased when shaded, as would be expected. Similarly, the levels of sugars (glucose, fructose, and sucrose) in the treated mature leaves increased in elevated CO2 and decreased with shade treatment. The levels of sugar in developing leaves were also measured and it was found that they mirrored this result even though they were not receiving the shade or elevated CO2 treatment. To investigate the effect of these treatments on global gene expression patterns, transcriptomics analysis was carried out using Affymetrix, 22K, and ATH1 arrays. Total RNA was extracted from the developing leaves after the mature leaves had received either the ambient control treatment, the elevated CO2 treatment, or the shade treatment, or both elevated CO2 and shade treatments for 2, 4, 12, 24, 48, or 96 h. The experiment was replicated four times. Two other experiments were also conducted, one to compare and contrast gene expression in response to plants grown at elevated CO2 and the other to look at the effect of these treatments on the mature leaf. The data were analysed and 915 genes from the untreated, signalled leaves were identified as having expression levels affected by the shade treatment. These genes were then compared with those whose transcript abundance was affected by the shade treatment in the mature treated leaves (1181 genes) and with 220 putative ‘stomatal signalling’ genes previously identified from studies of the yoda mutant. The results of these experiments and how they relate to environmental signalling are discussed, as well as possible mechanisms for systemic signalling.
The metabolism of cis-tramadol has been studied in human liver microsomes and in cDNA-expressed human cytochrome P-450 (CYP) isoforms. Human liver microsomes catalyzed the NADPH-dependent metabolism ...of tramadol to the two primary tramadol metabolites, namely, O-desmethyl-tramadol (metabolite M1) and N-desmethyl-tramadol (metabolite M2). In addition, tramadol was also metabolized to two minor secondary metabolites (each comprising < or =3.0% of total tramadol metabolism), namely, N,N-didesmethyl-tramadol (metabolite M3) and N,O-didesmethyl-tramadol (metabolite M5). Kinetic analysis revealed that multiple CYP enzymes were involved in the metabolism of tramadol to both M1 and M2. For the high-affinity enzymes involved in M1 and M2 formation, K(m) values were 116 and 1021 microM, respectively. Subsequent reaction phenotyping studies were performed with a tramadol substrate concentration of 250 microM. In studies with characterized human liver microsomal preparations, good correlations were observed between tramadol metabolism to M1 and M2 and enzymatic markers of CYP2D6 and CYP2B6, respectively. Tramadol was metabolized to M1 by cDNA-expressed CYP2D6 and to M2 by CYP2B6 and CYP3A4. Tramadol metabolism in human liver microsomes to M1 and M2 was markedly inhibited by the CYP2D6 inhibitor quinidine and the CYP3A4 inhibitor troleandomycin, respectively. In summary, this study demonstrates that cis-tramadol can be metabolized to tramadol metabolites M1, M2, M3, and M5 in human liver microsomal preparations. By kinetic analysis and the results of the reaction phenotyping studies, tramadol metabolism in human liver is catalyzed by multiple CYP isoforms. Hepatic CYP2D6 appears to be primarily responsible for M1 formation, whereas M2 formation is catalyzed by CYP2B6 and CYP3A4.
The current United States Environmental Protection Agency (EPA) classification of di(2-ethylhexyl)phthalate (DEHP) as a B2 “probable human” carcinogen is based on outdated information. New toxicology ...data and a considerable amount of new mechanistic evidence were used to reconsider the cancer classification of DEHP under EPA's proposed new cancer risk assessment guidelines. The total weight-of-evidence clearly indicates that DEHP is not genotoxic. In vivo administration of DEHP to rats and mice results in peroxisome proliferation in the liver, and there is strong evidence and scientific consensus that, in rodents, peroxisome proliferation is directly associated with the onset of liver cancer. Peroxisome proliferation is a transcription-mediated process that involves activation by the peroxisome proliferator of a nuclear receptor in rodent liver called the peroxisome proliferator-activated receptor (PPARα). The critical role of PPARα in peroxisomal proliferation and carcinogenicity in mice is clearly established by the lack of either response in mice genetically modified to remove the PPARα. Several mechanisms have been proposed to explain how, in rodents, peroxisome proliferation can lead to the formation of hepatocellular tumors. The general consensus of scientific opinion is that PPARα-induced mitogenesis and cell proliferation are probably the major mechanisms responsible for peroxisome proliferator-induced hepatocarcinogenesis in rodents. Oxidative stress appears to play a significant role in this increased cell proliferation. It triggers the release of TNFα by Kupffer cells, which in turn acts as a potent mitogen in hepatocytes. Rats and mice are uniquely responsive to the morphological, biochemical, and chronic carcinogenic effects of peroxisome proliferators, while guinea pigs, dogs, nonhuman primates, and humans are essentially nonresponsive or refractory; Syrian hamsters exhibit intermediate responsiveness. These differences are explained, in part, by marked interspecies variations in the expression of PPARα, with levels of expression in humans being only 1–10% of the levels found in rat and mouse liver. Recent studies of DEHP clearly indicate a nonlinear dose–response curve that strongly suggests the existence of a dose threshold below which tumors in rodents are not induced. Thus, the hepatocarcinogenic effects of DEHP in rodents result directly from the receptor-mediated, threshold-based mechanism of peroxisome proliferation, a well-understood process associated uniquely with rodents. Since humans are quite refractory to peroxisomal proliferation, even following exposure to potent proliferators such as hypolipidemic drugs, it is concluded that the hepatocarcinogenic response of rodents to DEHP is not relevant to human cancer risk at any anticipated exposure level. DEHP should be classified an unlikely human carcinogen with a margin of exposure (MOE) approach to risk assessment. The most appropriate and conservative point of reference for assessing MOEs should be 20 mg/kg/day, which is the mouse NOEL for peroxisome proliferation and increased liver weight. Exposure of the general human population to DEHP is approximately 30 μg/kg body wt/day, the major source being from residues in food. Higher exposures occur occupationally up to about 700 μg/kg body wt/day (mainly by inhalation) based on current workplace standards and through use of certain medical devices e.g., up to 457 μg/kg body wt/day for hemodialysis patients (intravenous), although these have little relevance because the routes of exposure bypass critical activation enzymes in the gastrointestinal tract.
1. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan®) was used to examine the induction of some selected rat hepatic cyto-chrome P450 (CYP) forms in vivo and ...in vitro using cultured precision-cut liver slices. 2. TaqMan primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1, CYP2B1/2, CYP3A1, CYP3A2 and CYP4A1 mRNAs. 3. To characterize the responsiveness of the rat CYP mRNA TaqMan primers and probe sets, rats were treated in vivo with a single intraperitoneal dose of 500 mg kg − 1 Aroclor 1254 (ARO) and with four daily oral doses of either 50 mg kg − 1 day − 1 dexamethasone (DEX) or 75 mg kg − 1 day − 1 methylclofenapate (MCP). Treatment with ARO produced 22 600-, 5480-, 648-, 52-, 47- and 9-fold increases in levels of CYP1A1, CYP2B1, CYP2B1/2, CYP1A2, CYP3A1 and CYP3A2 mRNA, respectively. DEX treatment produced 97-, 24-, 8- and 4-fold increases, respectively, in CYP3A1, CYP2B1, CYP2B1/2 and CYP3A2 mRNA levels, and MCP produced 339-, 126- and 25-fold increases, respectively, in CYP4A1, CYP2B1 and CYP2B1/2 mRNA levels. All three CYP inducers also increased microsomal CYP content and produced corresponding increases in CYP1A, CYP2B, CYP3A and CYP4A form marker enzyme activities. 4. Rat liver slices were cultured for 6 and 24 h in medium containing 0.1 µ M insulin and 0.1 µ M DEX, and also for 24 h in medium containing only 0.1 µ M insulin (DEX-free medium). Liver slices were cultured in control medium or in medium containing either 10 µ M β -naphthoflavone (BNF), 10 µ g ml − 1 ARO, 500 µ M sodium phenobarbitone (NaPB), 20 µ M pregnenolone-16 α -carbonitrile (PCN), 50 µ M Wy-14,643 (WY) or 50 µ M MCP. 5. With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all the CYP mRNAs studied was greater after 24- than after 6-h treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 h in liver slices cultured in DEX-free than in DEX-supplemented medium. 6. Treatment of liver slices with BNF and ARO for 24 h in DEX-free medium produced 21- and 35-fold increases, respectively, and 38- and 37-fold increases, respectively, in CYP1A1 and CYP1A2 mRNA levels. NaPB, PCN, WY and MCP did not increase either CYP1A1 or CYP1A2 mRNA levels. 7. After 24 h, levels of CYP2B1/2 mRNA were increased 18-, 20-, 9-, 16- and 13-fold by treatment with ARO, NaPB, PCN, WY and MCP, respectively. PCN also produced 56- and 4-fold increases, respectively, in CYP3A1 and CYP3A2 mRNA levels. 8. Treatment with WY and MCP for 24 h produced 437- and 186-fold increases, respectively, in levels of CYP4A1 mRNA. None of the other CYP inducers studied had any effect on CYP4A1 mRNA levels. 9. The results demonstrate the utility of cultured precision-cut liver slices as an in vitro model system to evaluate the effects of xenobiotics on rat CYP1A, CYP2B, CYP3A and CYP4A form mRNA levels.
1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products ...including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (± SEM) Km and Vmax of 93 ± 18 mm and 317 ± 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80µM ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r2 = 0.774) and phthalazine (r2 = 0.460). 5. The metabolism of 80µM ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with Ki of 2.3 and 1.9 µM, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H218O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H218O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.
A wide variety of chemicals have been shown to produce liver enlargement, peroxisome proliferation, and induction of peroxisomal and microsomal fatty acid-oxidizing enzyme activities in rats and ...mice. Moreover, certain peroxisome proliferators have been shown to increase the incidence of liver tumors in these two species. This review describes the characteristics of peroxisome proliferation in rodent liver and in vitro in primary hepatocyte cultures and gives examples of the range of different classes of chemicals that produce this effect. Mechanisms of initiation of peroxisome proliferation in rodent hepatocytes, including peroxisome proliferator-activated receptors, are also described. Peroxisome proliferators are not considered to be genotoxic carcinogens, and proposed mechanisms of liver tumor formation include induction of sustained oxidative stress, a role for enhanced cell replication, and the promotion of spontaneous preneoplastic lesions. Data are also presented on species differences in response and key issues concerning the risk assessment to humans of rodent liver peroxisome proliferators.
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