Jian-Dong Duan

Methyleugenol hepatocellular cancer initiating effects in rat liver.

Methyleugenol (MEG), a constituent of plants used in the human diet, is hepatocarcinogenic in rodents. In an experiment to elucidate its mode of action in rat liver, male F344 rats were administered MEG intragastrically at 3 doses per week for up to 16 weeks in an initiation phase, after which half the rats were fed 500 ppm phenobarbital (PB) in the diet to promote liver neoplasia and the other half were maintained on control diet for 24 weeks. At 8 and 16 week interim terminations, (32)P-nucleotide postlabeling assay revealed 3 adducts in livers of all MEG groups. The hepatocellular replicating fractions, measured by proliferating cell nuclear antigen immunohistochemistry, were doubled or more in all MEG groups. Hepatocellular altered foci, detected by glutathione S-transferase-placental type (π) immunohistochemistry, were present beginning with the high dose group at 8 weeks and extending to all MEG groups at 16 weeks. At the end of maintenance/promotion phase, the incidences, multiplicity and size of foci was similar between control and low dose groups, while those of mid and high dose groups were increased. Hepatocellular adenomas occurred in the mid and high dose groups, attaining higher multiplicity and size with PB. Thus, MEG had rapid initiating activity, reflecting the formation of DNA adducts and possibly cell proliferation.

Assessment of the Medicines Lidocaine, Prilocaine, and Their Metabolites, 2,6-Dimethylaniline and 2-Methylaniline, for DNA Adduct Formation in Rat Tissues

The local anesthetics lidocaine (lido) and prilocaine (prilo) are metabolized to their constituent aromatic amines 2,6-dimethylaniline (DMA, 2,6-xylidine) and 2-methylaniline (MA, o-toluidine), respectively, which are both tumorigenic in rats. The capacity of lido and prilo to form DNA adducts was assessed in major target tissues for aromatic amines in male F344 rats in comparison to equimolar doses of DMA and MA using the 32P-postlabeling assay. Direct reaction of putative DNA-reactive metabolites N-hydroxy-DMA and N-hydroxy-MA with isolated DNA yielded reference adducts. Rats were dosed by p.o. gavage with 0.5 mmol/kg b.wt. of each test substance or the vehicle either once or daily for 7 days. After repeat administrations of either prilo or lido, DNA adducts were detected in the liver and nasal mucosa. Urinary bladder DNA adducts were detected only in lido and DMA repeat dosed rats. Groups dosed with DMA or MA showed adducts in both single- and multiple-dose groups, except for the single-dose DMA liver and urinary bladder samples, which were below the level of detection. No DNA adducts were detected in any of the white blood cell samples under either dosing regimen. The lido- and prilo-DNA adducts detected were chromatographically indistinguishable from those formed either in DMA- or MA-dosed rats, respectively, or by chemical reaction of the corresponding N-hydroxy derivatives with DNA. Thus, lido and prilo can generate DNA adducts in rats via their aromatic amine metabolites, although at lower levels than equal molar quantities of their amine metabolites.

Furan induction of DNA cross-linking and strand breaks in turkey fetal liver in comparison to 1,3-propanediol

Furan, a food contaminant formed by heating, is hepatocarcinogenic to rats and mice. Conflicting genotoxicity data exist on furan and its metabolite, cis-2-butene-1,4-dial and there are few data for the target organ, the liver. We assessed the abilities of furan and, as a positive control, 1,3-propanediol (PDO), to cause DNA damage in the livers of turkey fetuses in ovo using the alkaline comet assay. Single injections of furan (2-20 μmoles) into turkey eggs, at 23 days of incubation, when the liver is well developed, reduced the %DNA in the comet tail (%DNA-CT) in hepatocytes isolated from fetuses 24 h later indicating DNA cross links. Treatment of the hepatocytes with proteinase K, digest DNA-protein cross links (DPXLs), increased the %DNA-CT compared to the corresponding controls, indicating the presence of DNA single or double stand breaks (SB). PDO showed little toxicity and was used at high doses (up to 300 μmoles/egg), where it induced DPXLs at about 20 times the furan dose. Thus, furan produced dose proportional reductions in %DNA-CT in turkey liver fetal hepatocytes indicating the presence of DPXLs and, after proteinase K treatment, an increase in %DNA-CT, indicating the presence of DNA single and/or double SB.

Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens.

Relationship of cellular topoisomerase IIα inhibition to cytotoxicity and published genotoxicity of fluoroquinolone antibiotics in V79 cells.

Fluoroquinolone (FQ) antibiotics are bacteriocidal through inhibition of the bacterial gyrase and at sufficient concentrations in vitro, they can inhibit the homologous eukaryotic topoisomerase (TOPO) II enzyme. FQ exert a variety of genotoxic effects in mammalian systems through mechanisms not yet established, but which are postulated to involve inhibition of TOPO II enzymes. To assess the relationship of inhibition of cell nuclear TOPO II to cytotoxicity and reported genotoxicity, two FQ, clinafloxacin (CLFX) and lomefloxacin (LOFX), having available genotoxicity data showing substantial differences with CLFX being more potent than LOFX, were selected for study. The relative inhibitory activities of these FQ on nuclear TOPO IIα in cultured Chinese hamster lung fibroblasts (V79 cells) over dose ranges and at equimolar concentrations were assessed by measuring nuclear stabilized cleavage complexes of TOPO IIα-DNA. Cytotoxicity was measured by relative cell counts. Both FQ inhibited V79 cell nuclear TOPO IIα. The lowest-observed-adverse-effect levels for TOPO IIα inhibition were 55 μM for CLFX, and 516 μM for LOFX. The no-observed-adverse-effect-levels were 41 μM for CLFX, and 258 μM for LOFX. At equimolar concentrations (175 μM), CLFX was more potent than LOFX. Likewise, CLFX was more cytotoxic than LOFX. Thus, the two FQ, inhibited TOPO IIα in intact V79 cells, differed in their potencies and exhibited no-observed-adverse-effect levels. These findings are in concordance with published genotoxicity data and observed cytotoxicity.

A no observed adverse effect level for DNA adduct formation in rat liver with prolonged dosing of the hepatocarcinogen 2-acetylaminofluorene

We have previously reported that the DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF) produced in rat liver several key effects that were less than linear over a range of repeat doses, and that at low cumulative doses, no-adverse-effect-levels (NOAEL) were observed for several effects including hepatocellular cytotoxicity, enhanced cell proliferation, induction of preneoplastic foci and promotable liver neoplasms, although DNA adducts were still formed at dosages below the lowest NOAEL of 28 mg kg−1 cumulative dose for these other effects. This report details two further dose-effect studies at lower repeat doses than those previously used as well as identification of the specific types of DNA adducts formed. AAF was administered orally to male F344 rats by gavage at repeat dosages which in one experiment (A) ranged from 0.01 to 2.24 mg kg−1 bw per day, 7 days per week for 12 weeks followed by recovery for 4 weeks, and in a second (B) at lower dosages of 0.0026 or 0.026 mg kg−1 bw per day 3 days per week for 16 weeks. Initially the nonacetylated guanine adduct, N-(deoxyguanosine-8-yl)-aminofluorene, predominated. With continued dosing, the pattern of adducts changed such that by 4 weeks more acetylated, N-(deoxyguanosine-N2-yl)-AAF and N-(deoxyguanosine-8-yl)-AAF, adducts were present. In experiment A, total adducts reached a maximum by 12 weeks with levels of 6.0 adducts per 108 nucleotides at the lowest dosage. In experiment B, the total DNA adducts at the lowest dosage was below the limit of detection at 12 weeks, and at 0.6 in 108 nucleotides at 16 weeks, levels within the background range of 1.0–3.1 per 108 nucleotides. Thus, the cumulative dose of 0.125 mg kg−1 bw over 16 weeks was a NOAEL for adducts and hence would be predicted to be a threshold for hepatocarcinogenicity.

In Vivo Genotoxicity of Estragole in Male F344 Rats

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio‐activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8‐oxoguanine‐DNA‐N‐glycosylase (hOGG1) and EndonucleaseIII (EndoIII)‐modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven‐week‐old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole‐induced dose‐dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non‐target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole‐dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical‐specific interaction and is also mediated by oxidative species. Environ. Mol. Mutagen. 56:356–365, 2015.

Hepatocellular proliferation and hepatocarcinogen bioactivation in mice with diet-induced fatty liver and obesity

Human liver cancer is in part associated with obesity and related metabolic diseases. The present study was undertaken in a mouse model of diet-induced obesity (DIO) and hepatic steatosis, conditions which can be associated with hepatic neoplasia, to determine whether the rates of cell proliferation or hepatocarcinogen bioactivation were altered in ways which could facilitate hepatocarcinogenesis. DIO mice were generated by feeding C57BL/6 (B6) male mice a high-fat diet beginning at 4 weeks of age; age-matched conventional lean (LEAN) B6 mice fed a low fat diet (10% Kcal from fat) were used for comparison. Groups of 28 week old DIO and LEAN mice were dosed with the bioactivation-dependent DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF), at 2.24 or 22.4 mg/kg, given by gavage 3 times per week for 31 days, or received no treatment (DIO and LEAN control groups). Compared with the LEAN control group, the DIO control group had a higher mean body weight (16.5 g), higher mean absolute (1.4 g) and mean relative (25.5%) liver weights, higher (394%) liver triglyceride concentrations, and an increased incidence and severity of hepatocellular steatosis at the end of the dosing phase. The DIO control group also had a higher mean hepatocellular replicating fraction (31% increase, determined by proliferating cell nuclear antigen immunohistochemistry). Hepatocarcinogen bioactivation, based on formation of AAF DNA adducts as measured by nucleotide (32)P-postlabeling, was similar in both DIO and LEAN AAF-dosed groups. Thus, hepatocellular proliferation, but not hepatocarcinogen bioactivation, was identified as an alteration in livers of DIO mice which could contribute to their susceptibility to hepatocarcinogenesis.

Inhibition by acetaminophen of neoplastic initiation elicited in rat liver by the DNA-reactive hepatocarcinogen N-acetyl-2-aminofluorene.

Acetaminophen, a monocyclic phenolic compound and analgesic, when fed at 8900 p.p.m. in the diet, was reported to inhibit the hepatocarcinogenicity in rats of the aromatic amine proximate carcinogen N-hydroxy-N-acetyl-2-aminofluorene. To elucidate the mechanism(s) of this anticarcinogenicity, the present study examined whether acetaminophen at lower doses has the ability to inhibit the initiating effects in the rat liver of the precursor hepatocarcinogen N-acetyl-2-aminofluorene. Male F344 rats were allocated to six groups, which were maintained under reverse light cycle conditions to assure acetaminophen ingestion at the time of N-acetyl-2-aminofluorene administration during the dark phase, which was imposed from 07.00 to 19.00 h. Group 1 served as vehicle control (0.5% carboxymethylcellulose) for N-acetyl-2-aminofluorene, which was administered intragastrically 3 days per week at 2.6 mg/kg for 8 weeks (group 4) to achieve initiation. Acetaminophen was given in the diet either alone at 2400 or 4800 p.p.m. for 9 weeks (groups 2 and 3), or with N-acetyl-2-aminofluorene (groups 5 and 6), starting 1 week before N-acetyl-2-aminofluorene administration. Acetaminophen blood levels were about 1 and 4 μg/ml at the two dietary concentrations. N-acetyl-2-aminofluorene induced hepatocellular preneoplastic lesions measured as hepatocellular altered foci expressing glutathione S-transferase-P, reflecting initiation. Induced foci were reduced with administration of both concentrations of acetaminophen. Acetaminophen by itself produced no DNA adducts nor did it alter the high formation of N-acetyl-2-aminofluorene–DNA adducts, about 200 in 108 nucleotides, measured by nucleotide postlabeling. Acetaminophen did not affect background liver cell proliferation, but significantly reduced N-acetyl-2-aminofluorene-induced increased proliferation measured by proliferating cell nuclear antigen immunostaining. Thus, acetaminophen effectively protected hepatocytes from the initiating effects of N-acetyl-2-aminofluorene, possibly through a cytoprotective effect resulting from slowing the rate of induced cell turnover.

Chicken egg fetal liver DNA and histopathologic effects of structurally diverse carcinogens and non-carcinogens

Chicken egg fetal livers were evaluated for histopathological changes produced by four genotoxic hepatocarcinogens: 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), diethylnitrosamine (DEN); four structurally related non- or weakly- carcinogenic comparators: fluorene (FLU), aflatoxin B2 (AFB2), benzo[e]pyrene (BeP), N-nitrosodiethanolamine (NDELA); two epigenetic hepatocarcinogens: clofibric acid (CFA), phenobarbital (PB); and the non-carcinogen, D-mannitol (MAN). CFA, PB and MAN were also assessed for formation of DNA adducts using the 32P nucleotide postlabeling (NPL) assay and for DNA breaks using the comet assay. CFA was also assessed in enhanced comet assay for oxidative DNA damage induction. Eggs were dosed on days 9- 11 of incubation. For genotoxicity evaluation, livers were collected 3h after the last dose. Liver qualitative histopathology assessment was performed on days 12 and 18 of incubation. CFA was negative for DNA adducts but yielded clear evidence of DNA breaks due to oxidative stress. PB and MAN produced no DNA adducts or breaks. Liver to body weight ratios were not affected in most groups, but were decreased in DEN groups, and increased after PB dosing. Livers from control groups, FLU, AFB2, BeP, NDELA, CFA, and MAN groups, displayed a typical hepatocellular trabecular pattern at both time points. In contrast, the four genotoxic carcinogens induced time- and dose- related interference with fetal liver cell processes of proliferation, migration and differentiation, leading to hepatocellular and cholangiocellular pleomorphic dysplasia and re-(de-) differentiation with distortion of the trabecular pattern. In addition, dosing with the high dose of DEN produced gallbladder agenesis. PB induced hepatocellular hypertrophy, interference with migration, expressed as distortion of the trabecular pattern, and a moderate cholangiocellular dysplasia. In summary, histopathological analysis of chicken fetal livers revealed developmental anomalies, as well as genotoxicity-induced and, in the case of PB, adaptive morphological changes. Thus, the model provides histopathological outcomes of molecular effects.