Differentiated genotoxic response of carcinogenic and non-carcinogenic benzacridines and metabolites in rat hepatoma cells

Two closely related hepatoma cell lines were examined for theirgenotoxic response to benzacridines and their metabolites bythe appearance of alkaline labile DNA sites: H₅, a dedifferentiatedline expressing cytochrome P-448-dependent mono-oxygenase(s);and HF_1–4, a differentiated line expressing cytochromeP-450-dependent monooxygenase(s). The parent heterocycles hadno effect on both cell lines. In contrast to the 3, 4-dihydrodiolof benz[c]acridine the 3, 4-dihydrodiol of benz[a]acridine inducedno DNA strand breaks in both cell lines. All diol epoxides,however, induced DNA-damage in both cell lines, the syn derivativesin the same order of magnitude as the dihydrodiol of benz[c]acridine.The antidiol epoxides (epoxide group on the opposite side tothe benzylic hydroxyl group) were the most potent to induceDNA-single strand breaks. The diol epoxide of benz[c]acridinewas three times more efficient in HF_1–4 than in H5, whereasfor the diol epoxide of benz[a]acridine, the reverse was true.The results indicate that benz[c]acridine-3, 4-diol is oxidizedto metabolites which can induce DNA-damage. This is consistentwith the hypothesis that the benz[a]acridine and derivativesare not easily metabolized to active mutagens but more likelyare converted to inactive metabolites, possibly via N-oxidation.This is illustrated with 3, 4-diol-1-2 anti-diol epoxide ofbenz[a]acridine which is inactivated in cell line HF_1–4due to the reactivity of the epoxide ring in the bay region.Since all diol epoxides show similar activity in both hepatomacell lines, they are of great interest because of their abilityto detect DNA-damaging agents and to analyse their metabolicactivation and mechanism of action.     

Specificity of rat liver cytochrome P-450 isozymes in the mutagenic activation of benzo[a]pyrene, aromatic amines and aflatoxin B1

The ability of three purified forms of rat liver cytochromeP-450 to metabolically activate benzo[a]pyrene, trans-benzo-[a]pyrene-7,8-dihydrodiol,2-aminofluorene, afiatoxin B₁, dimethylnitrosamine, and a pyrolysisproduct of tryptophan(3-amino-l-methyl-5H-pyrido(4,3-b)indole)(Trp-P-2) to muta-genic products was examined using Salmonellatyphimurium strains TA98 and G46 in a reconstituted monooxygenasesystem. The isozymes examined were cytochrome P-450-PB (themajor phenobarbital inducible form), and the two major 3-MCinducible forms (cytochromes P-448₅₂ and P-448₅₅). CytochromesP-448₅₂ and P-448₅₅ preferentially metabolize 2-aminofluoreneand Trp-P-2 to mutagenic products. However, only cytochromeP-448₅₅ metabolizes benzo[a]pyrene and its 7,8-dihydrodiol derivativeto mutagenic products. Both cytochrome P-448₅₂ and P-448₅₅ metabolizeafiatoxin B, to mutagenic products at a much faster rate thancytochrome P-450-PB. Dimethylnitrosamine was not activated byany of the isozymes tested.     

Immunochemical study on the contributions of two molecular species of cytochrome P-450 in mutagenesis by selected aminoazo dyes

The relative contributions of two species of cytochrome P-450,the major cytochrome P-450 components of liver microsomes ofphenobarbital-treated rats (PB-P-450) and of 3-methylcholanthrene-treatedrats (MC-P-448), in the mutagenic activation of 3'-methyl-N,N-dimethyl-4-aminoazobenzene(3'-Me-DAB) and its eight metabolites were studied in the Salmonellaassay system using specific antibodies and inhibitors. The antibodyagainst MC-P-448 considerably inhibited the mutagenicities of3'-Me-DAB, 3'-CH₂OH-DAB, their three N-demethylated compounds,3'-CHO-DAB, and 3'-Me-4'-OH-DAB in strain TA98, whereas theantibody against PB-P-450 inhibited their mutagenicities <29%.In contrast, the antibody against MC-P-448 caused no or slight(29%) inhibition of the mutagenicities of 3'-hydoxymethyl-N-methyl-4-aminoazobenzene(3'-CH₂OH-MAB) and 3'-COOH-DAB. However, the mutagenicitiesof both compounds were considerably inhibited by 7,8-benzo-flavone,like those of other seven aminoazo dyes. These results demonstratethat rat liver cytochrome P-450, especially MC-P-448, is involvedin mutagenic activation of the aminoazo dyes. Participationof a second form of cytochrome P-448 in mutagenic activationof 3'-CH₂OH-MAB and 3'-COOH-DAB is discussed.     

The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol

The effects of ethanol on the metabolism of nitrosamines byrat liver microsomes have been studied. Treatment of rats with10 or 15% ethanol in drinking water for 3 days causes a 4- to5-fold enhancement in microsomal N-nitrosodimethylamine demethylase(NDMAd) activity and a 40–60% increase in gross P-450content. The enhancement is mainly due to the induction of alow K_m form (K_m = 0.07 mM) of NDMAd. The treatment induces proteinspecies with molecular weights between 50 000 and 52 000, someof which are believed to be P-450 isozymes with high affinityto NDMA. In addition to NDMA, treatment with ethanol also enhancesthe metabolism of N-nitroso-N-methylethylamine, N-nitrosomethylamline,and N-nitroso-N-methylbenzylamine. When added to the incubationmixture, ethanol and its homologs inhibit the demethylationof these nitrosamines by microsomes. Ethanol is a competitiveinhibitor of the low K_m NDMAd with a K_i of 0.31 mM and is lesseffective in inhibiting the metabolism of more lipophilic nitrosamines.     

Inhibition of aflatoxin B1 -hepatocarcinogenesis in rats by {beta}-naphthoflavone

Effects of ß-naphthonflavoe (ßNF) on theactivity of hepatic microsomal aflatoxin B₁ (AFB₁)-4-hydroxylase- the cytochrome P-450-dependent enzyme system which catalyzesthe metabolism of AFB₁ to AFM₁ - and on AFB₁ induced in vivohepatocarcinogenesis were investigated in weanling male Fischerrats. A single i.p. injection of ßNF in doses of 20mg/kg and 150 mg/kg induced AFB₁ -4-hydroxylase 3- and 4-fold,respectively, 48 h post injection. Feeding of diet containing0.01% ßNF for a period of 9-weeks induced AFB₁ 2-fold.AFB₁, given by intubatlon in a dose of 25 µg five times/weekfor 8 weeks, produced 42 weeks later a 100% incidence of liverlesions (neoplastic foci, nodules or tumors), but feeding ßNFin diet at a con centration of 0.015% for one week prior toand during the 8 weeks of AFB treatment inhibited AFB₁ hepatocarcinogenesis by -7%. These results are in accord with the suggestionthat AFB₁ induction may be associated with the inhibition ofAFB₁ carcinogenesis, possibly occurring as a consequence ofaccelerated detoxi-fication of AFB₁ via its conversion to AFM₁     

Metabolism of aflatoxin B1 in the bovine olfactory mucosa

Carcinomas of the ethmoidal region of the nose are observedrelatively frequently in cattle in several countries in tropicaland subtropical latitudes. Viruses have been implicated as causativeagents, but it has been observed that affected animals sometimessuffer from aflatoxicosis, and a role of aflatoxin B₁ (AFB₁)in the aetiology has also been proposed. We have examined whetherthe bovine nasal olfactory mucosa has a capacity to metabolizeAFB₁. The contents of cytochrome P–450 and cytochromeb₅, and the NADPH cytochrome c reductase activity in the nasalolfactory mucosa have also been determined. Comparative experimentshave been performed with the liver. Incubations with ³H-labelledAFB₁ showed that the nasal olfactory mucosa has a much highercapacity than the liver to form lipid-soluble, water-solubleand tissue-bound AFB₁-metabolites. High-resolution microautoradiographyshowed a strong localization of tissue-bound metabolites inthe sustentacular cells in the apical portion of the olfactorysurface epithelium and in Bowman's glands in the olfactory laminapropria mucosae. Especially in the sustentacular cells the labellingwas preferentially located in the nuclei of the cells. Liquidchromatography of chloroform extracts of the nasal olfactorymucosa and the liver incubated with ³H-AFB₁ showed formationof several metabolites. The dominating peak in both tissueswas aflatoxin M₁ (AFM₁). However, the amount of AFM₁ was higherin the nasal olfactory mucosa than in the liver, and the amountsand proportions of several other metabolites also differed markedlybetween the two tissues. The level of cytochrome P-450 in thenasal olfactory mucosa was found to be about one quarter ofthat in the liver, but the NADPH cytochrome c reductase activitywas much higher in the nasal olfactory mucosa than in the liver.In addition, the cytochrome b₅: cytochrome P-450 ratio was higherin the nasal olfactory mucosa than in the liver. The highermetabolism of AFB₁ in the nasal olfactory mucosa than in theliver may be related to differences in the cytochrome P-450isoenzyme profile. In addition, the microsomal electron transportto cytochrome P-450 may be facilitated by the high reductase:cytochrome P–450 ratio and the high cytochrome b₅: cytochromeP–450 ratio in the nasal olfactory mucosa. It is consideredthat the results of the present study strengthen the hypothesisthat exposure of AFB₁-contaminated feed may be an importantaetiological factor in the development of nasal tumours in cattle.     

Different mechanisms are involved in DNA damage, bacterial mutagenicity and cytotoxicity induced by 1,2-dibromo-3-chloropropane in suspensions of rat liver cells

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measuredby an automated alkaline elution method, in suspensions of ratliver parenchymal cells at low concentrations (1–10 µM).At much higher concentrations (0.5–2.5 mM), DBCP was metabolizedto products that were mutagenic to Salmonella typhimurium TA100co-incubated with the liver cells. At these higher concentrationsa marked depletion of cellular glutathione was seen and at 2.5mM DBCP was cytotoxic. Perdeuterated DBCP (D₅DBCP) caused lessDNA damage in the liver cells than DBCP, most likely becauseof decrease in cytochrome P-450 dependent metabolism. A morepronounced decrease in mutagenicity occurred with (D₅DBCP) comparedto DBCP, whereas the two compounds were equally cytotoxic. Preincubationof the liver cells with diethylmaleate or buthionine sulfoximine,to lower cellular levels of glutathione, decreased DBCP inducedDNA damage. The decrease in DNA damage was proportional to thedecrease in cellular glutathione levels. In contrast, diethylmaleate enhanced DBCP-induced bacterial mutagenicity and cellularcytotoxicity. The cytotoxic effect could be partly blocked byaddition of ascorbate. From the data presented we suggest that:(i) cytochrome P-450 dependent oxidation as well as glutathioneconjugation are involved in DBCP in duced DNA damage, (ii) cytochromeP-450 dependent oxidation leads to formation of products mutagenicto bacteria and (iii) the cytotoxicity induced by DBCP in theliver cells in vitro is caused by oxidative damage followingglutathione depletion and/or direct membrane damage.     

Species, sex and organ differences in induction of a cytochrome P-450 isozyme responsible for carcinogen activation: effects of dietary hepatocarcinogenic tryptophan pyrolysate components in mice and rats

Both sexes of BALB/cxDBA/2 F₁ mice and F344 rats were treatedfor 1 week with a diet containing 0.02% of hepatocarcinogenictryptophan pyrolysate component (Trp P-1 or Trp P-2), and changesin the carcinogen activation enzyme activity in various organswere examined comparatively using a mutation test with Salmonellatyphimurium TA98 as a tester bacterium. Hepatic enzymes fromuntreated mice and rats showed a definite catalytic activityfor mutagenic activations of Trp P-1 and Trp P-2, whereas theactivities of other organs —such as lung, kidney, smallintestine and colon—were undetectable or very low. Inboth mice and rats either the Trp P-1 or Trp P-2 feeding resultedin induction of cytochrome P-450 isozyme(s), which could mediatein the liver but not in other organs the mutagenic activationof the carcinogen itself. As to the sex difference, the inductionof the activation enzyme(s) was greater in the female animalsthan in the males. Species difference in the activity of hepaticenzymes catalyzing the Trp P-1 and Trp P-2 mutageneses was alsoobserved in animals treated with the basal diet; the activitywas higher in mice than in the sex-matched rats (Trp P-1, {smalltilde}1.5-fold; Trp P-2, {small tilde}7-fold). When diet containingTrp P-1 or Trp P-2 was fed for 1 week, the activity of the ratliver for Trp P-1 mutagenesis was of a level similar to thatof the sex-matched mice, but for Trp P-2 mutagenesis it wasless than half that in the mice. The induced hepatic enzymesin mice and rats were suggested to be 3-methylcholanthrene-induciblecytochrome P-448 isozymes as determined by mutation tests withTrp P-1, Trp P-2 and two other substrates and by immunochemicalanalyses of rat hepatic cytochrome P-450 using monoclonal antibodiesagainst rat cytochrome P-448 isozymes. These results indicatethat a form of cytochrome P-450 responsible for activation ofTrp P-1 and Trp P-2 is inducible by dietary treatment of miceor rats with these carcinogens and that the amount of the cytochromeP-450, including resident and induced forms, is related to thespecies, sex and organ differences in their carcinogenic susceptibilityto these chemicals.     

Effects of sex difference, gonadectomy and estradiol on N-ethyl-N-nitrosourea-induced trigeminal nerve tumors in rats

The occurrence of trigeminal nerve tumors (TNTs) induced byneonatal administration of N-ethyl-N-nitrosourea (ENU) in WF? LE F₁ (F₁ rats was studied with special reference to sex difference,effect of gonadectomy and estradiol (E₂) administration. Experimentalgroups 1–6 were treated with 40 mg ENU/kg of body weightneonatally. They consisted of male, female, castrated male,ovariectomized female, E₂ pellet (0.1 mg, s.c.) supplementedand gonadectomized male and female rats respectively. Rats ofgroups 7–12 served as the respective controls withoutENU. All the rats were killed at 8 months of age. Levels ofserum E₂ and E₂ receptor (ER) of the TNTs were also examined.It was noted that the incidence of TNT was higher in males (79%)than in females (48%, P < 0.05) and did not change by castrationin males (91%) but increased in ovariectomized female rats (74%,P < 0.05). Administration of E₂ followed by gonadectomy inhibitedthe occurrence of TNTs in male rats (59%) but not in femalerats (60%). No TNT was observed in any control groups. Kidneytumors were the second most frequent tumors next to nervoussystem tumors in the present experiment. The incidence of kidneytumors was much higher in females (38%) than in males (4%, P< 0.05) and decreased by ovariectomy, whereas it increasedin male rats by E₂ administration. ER levels of TNTs and trigeminalnerve tissue were < 1 fmol/mg protein. These results suggestthat in rats treated with ENU neonatally, E₂ has an inhibitoryeffect on the induction of TNTs but may not be regulated throughER. E₂ also shows a promoting effect on kidney tumorigenesis.     

Apoptotic and anti-proliferative effects of fumonisin B1 in human keratinocytes, fibroblasts, esophageal epithelial cells and hepatoma cells

Fumonisin B₁ is associated with various animal and human carcinomasand toxicoses, including leukoencelphalomalacia, hepatocarcinoma,pulmonary edema and esophageal carcinoma. We have examined thecellular effects of fumonisin B₁ in vitro using cellular modelsystems relevant to potential human target tissues. Althoughfumonisin B₁ has been described as a mitogen in Swiss 3T3 cellsbased on stimulation of [³H]thymidine incorporation, in thecurrent work it was found that fumonisin B₁ inhibited incorporationof [³H]thymidine by cultured neonatal human keratinocytes andHepG2 human hepatocarcinoma cells at 10^–7 and 10^–4M respectively. Fumonisin B₁ also inhibited clonal expansionof normal human keratinocytes and HET-1A human esophageal epithelialcells at 10^–5 M and growth in mass culture of normal humanfibroblasts at 10^–7 M. The clonogenicity of normal humankeratinocytes decreased to 45.5% of controls afterexposure to10^–4 M fumonisin B₁ for 2 days. However, no differencesin the cell cycle distribution of cultured keratinocytes wasnoted after exposure to 10^–5 M fumonisin B₁ for 40 h.Theviability of normal human keratinocytes and HET-1A cellsdecreased as a result of fumonisin B₁ treatment, as determinedby a fluorescein diacetate/propidium iodide flow cytometriccell viability assay. Fumonisin B₁-treated keratinocytes releasednucleosomal DNA fragments into the medium 2–3 days afterexposure to 10^–4 M fumonisin B₁ and increased DNA strandbreaks were detected in attached keratinocytes exposed to 0–10^–4M fumonisin B₁ using a terminal deoxy-nucleotidyl transferase-basedimmunochemical assay system. Furthermore, fumonisin B₁-treatedkeratinocytes and HET-1A cells developed morphological featuresconsistent with apoptosis, as determined by phase contrast microscopy,fluorescent microscopy of acridine orange stained cells andelectron microscopy. These results are consistent with the occurrenceof fumonisin B₁-mediated apoptosis in vitro.     

Hepatic cytochrome P-450 isozyme(s) induced by dietary carcinogenic aromatic amines preferentially in female mice of DBA/2 and other strains

DBA/2, BALB/c or (BALB/cxDBA/2)F₁ (CDF₁) mice of both sexeswere treated for 1 week with a dietary hepatocarcinogenic tryptophanpyrolysate component (Trp P-1 or Trp P-2), and the activityof hepatic microsomal enzyme(s) for mutagenk activations ofTrp P-1 and Trp P-2 were assessed by means of a mutation testwith Salmonella typhimurium TA98. In both Ah-responsive (BALB/cand CDF₁) and Ah-nonresponsive (DBA/2) mice, the dietary treatmentwith Trp P-l or Trp P-2 resulted in a significant increase ofthe enzyme activity for mutagenic activations of Trp P-1 andTrp P-2 in females but not in males, except the case of maleBALB/c mice treated with dietary Trp P-1 Also induction of enzyme(s)in female mice was suppressed by an administration of testosterone.The induced hepatic microsomal enzyme(s) was demonstrated tobe cytochrome P-450 isozyme(s) (mol. wt of 55 000 daltons) byimmunoblots with use of an anti-rat cytochrome P-448 monoclonalantibody and by selective inhibition of the activity by additionof 7,8-benzoflavone into the mutation assay system. These findingsindicate that carcinogic aromatic amines such as Trp P-1 andTrp P-2 are able to induce hepatic cytochrome P-450 isozyme(s)not only in Ah-responsive mice (BALB/c and CDF₁) but also inAh-nonresponsive DBA/2 mice and that the cytochrome P-450 inductionis controlled by androgen(s).     

Induction of sister chromatid exchanges in cultured adult rat hepatocytes by directly and indirectly acting mutagens/carcinogens

Primary cultures of adult rat hepatocytes were tested for theirsuitability to assess sister chromatid exchange (SCE)-inducingDNA damage produced by both directly and indirectly acting mutagens/carcinogens.Compared to other genotoxicity assay systems which utilize themetabolizing activity of liver microsomes, this system is atleast 1–2 orders of magnitude more sensitive. The approximatedrug concentrations leading to a doubling of control SCE levelswere 2.5x10^–4 M for cyclophosphamide, 4.5x10^–5 Mfor dimethylnitrosamine, 2.5x10^–6 M for N-methyl-N-nitro-N-nitrosoguanidine,2x10^–10 M for aflatoxin B₁ (AFB₁) and 30 mJ for u.v. Themost potent inducer of SCE proved to be AFB₁, leading to a significantlyelevated level of exchanges at a concentration of 10^–12M. The increased background SCE levels observed (0.75 SCE/chromosome)appears to reflect the sensitivity of hepatocytes to SCE-inducingDNA damage resulting from the dietary intake of mutagenic/carcinogeniccompounds. In view of the high sensitivity and versatility ofthis genotoxicity assay system, it will be of use for the detectionof the low levels of mutagenic/carcinogenic compounds foundin the environment.     

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide(N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes(PMNL), chiefly neutrophils, elicited with intraperitoneal injectionsof proteose peptone, was examined. At 10⁶ PMNL/ml in media containinghalide (X^–), 0.14 M Cl^– ± 0.1 mM Br^–(without Ca⁺⁺ and Mg⁺⁺), addition of 10 nM phorbol myristateacetate (PMA) resulted in generation of superoxide anion andH₂O₂. Subsequent cetyltrimethylammonium Cl^– (Cetac) additionat 0.002% effected myeloperoxidase (MPO) activity release. PMNLtreated with PMA and/or Cetac did not metabolize N-OH-2-FAA(30 µM). However, 1–2 pulses of H₂O₂ (50 µM)after Cetac addition resulted in oxidation of N-OH-2-FAA toN-acetoxy-2-FAA (<0.5 µM) and 2-nitrosofluorene (2-NOF)(1–2 µM). In the presence of Br^– 2-NOF wasincreased (3–5 µM). The results are consistent withoxidation of N-OH-2-FAA by MPO/H₂O₂ and MPO/H₂O₂/X^– viatwo pathways: one electron oxidation leading to N-acetoxy-2-FAAand 2-NOF, and X^–-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA(10 µM) incubated with PMNL under similar conditions wasconverted non-enzymatically to 4-OH-2-FAA (5 µM) and enzymaticallyto N-OH-2-FAA (3 µM). In the presence of H₂O₂, smalleramounts of these products were formed. Formation of N-OH-2-FAAwas prevented by paraoxon (0.1 mM) suggesting O-deacetylaseactivity. However, accountability for N-acetoxy-2-FAA decreasedwith time, presumably because of binding to cellular macromolecules.With H₂O₂ addition, 2-NOF (10 µM) was converted to 0.5or 0.25 µM 2-nitrofluorene by active PMNL or heat-inactivatedcell lysates, respectively. Low recoveries of 2-NOF were alsoattributed to binding. The results suggest that PMNL may beinvolved in activation of the carcinogenic N-arylhydroxamicacids in vivo.     

Metabolic inactivation of N-nitrosamines by cytochrome P-450 in vitro and in vivo

Nitrite was formed on incubation of N-nitrosamines with both microsomal systems and a reconstituted system consisting of cytochrome P-450 and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH was omitted. Various inhibitors of the microsomal monooxygenase decreased nitrite generation. Furthermore, nitrite and a substantially higher amount of nitrate could be found in the urine of rats given N-nitrosodiphenylamine. Diphenylamine was also detected. From in-vitro studies, it is concluded that denitrosation of N-nitrosamines is a cytochrome P-450-dependent process, which also occurs in vivo.     

Phosphorylation of cytochrome-P-450-dependent monooxygenase components

Most chemical carcinogens require activation by polysubstratemonooxygenase. The phosphorylation of essential components ofthis cytochrome P-450 monooxygenase system, isolated from rabbitliver microsomes, cytochrome P-450 (LM₂) and cytochrome reductase,was tested using two different protein kinases. One of the kinases,a cyclic AMP-independent phosvitin kinase (kinase P), was inactivein all systems tested. However, the catalytic subunit of a cyclicAMP-dependent protein kinase (kinase C) catalyzed phosphorylgroup transfer to both proteins, but to different extents. CytochromeP-450 was phosphorylated when added as sole component and alsowhen in the presence of P-450 reductase and phosphatidylcholine.In contrast, the weak phosphorylation of P-450 reductase wasreduced considerably in a complete reconstituted system containingP-450 and phosphatidylcholine. The inclusion of kinase P didnot alter these results which excludes the possibility thatthese kinases participate in a sequential phosphorylation mechanism.The monooxygenase constituents themselves were without kinaseactivity. When hepatic microsomes were isolated in presenceof the phosphatase inhibitor sodium fluoride no significantchange in monooxygenase (7-ethoxycoumarin O-deethylation) activitywas observed, whilst after preincubation with either acid oralkaline phosphatase a significant reduction in monooxygenaseactivity was measured. Thus, cytochrome P-450 (LM₂) is phosphorylatableby protein kinase C and the catalytic activity of polysubstratemonooxygenase decreases after preincubation of microsomes withphosphatases.     

Potentiation of cytotoxicity by 3-aminobenzamide in DNA repair-deficient human tumor cell lines following exposure to methylating agents or anti-neoplastic drugs

We studied the potentiation by 3-aminobenzamide (3AB) of killingof nine human cell lines exposed to alkylating agents. Celllines included normal, transformed and DNA repairproficientand -deficient pbenotypes. 3AB potentiated cell killing by themethylating agents methyhnethanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in all lines tested. The degree of potentiation rangedfrom 1.7- to 3.8-fold, based on the LD₉₉. The average potentiationobserved with MMS (2.7-fold) was greater than with MNNG (2.2-fold).On average the potentiation of MMS and MNNG killing of repair-deficientMer^– lines (2.4-fold) was similar to that of repair-proficientMer⁺ lines. The degree of 3AB potentiation of MNNG killing (2.0-fold)was similar in Mer⁺ Rem^– lines and in Mer⁺ Rem⁺ lines.Mer⁺ Rem⁺, Mer⁺ Rem^–, Mer^– Rem⁺, and Mer^–Rem^– strains all appeared proficient in a 3AB-sensitiveDNA repair pathway. Within experimental error, 20 mM 3AB didnot inhibit the removal of the MNNG-induced methylpurines 7-methylguanine,O⁶-methylguanine and 3-methyladenine from the DNA of repair-proficientMer₊ Rem⁺ HT29 cells, consistent with evidence that 3AB inhibitsthe ligation step of excision repair. 3AB potentiated cell killingby the bifunctional alkylating agents 1-(2-chlorethyl)-1-nitrosoureaor busulfan, two antineoplastic drugs, by only 0.9- to 1.5-fold.These drugs therefore produce DNA damage which is not efficientlyrepaired by the pathways that repair methylated bases.     

Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays

We have demonstrated that the human cytochrome P₁-450 gene canbe transfected into the AHH-1 human lymphoblastoid cell lineusing the pHEBo vector and hygromycin selection. The transfectedgene was expressed when regulatory sequences derived from theherpes simplex virus thymidine kinase gene were incorporatedin appropriate orientations. Gene expression was monitored atthe enzyme level using assays for 7-ethoxyresorufin deethylase,7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase activities.Bulk transformed cell populations had 2- to 3-fold more of theseenzyme activities compared with control populations. Subclonesof the bulk population expressing still higher levels of 7-ethoxyresorufindeethylase activity were also obtained. Expression of the transfectedcytochrome P₁-450 gene was stable for 20–30 days in thepresence of hygromycin B. The transformed cell populations werefound to be suitable for use in gene locus mutation assays andthe mutagenicity of aflatoxin-B₁ and 2-acetylaminofluorene (AAF)were examined. Aflatoxin-B₁ was found to be 2–3 timesmore mutagenic to cells bearing the transfected cytochrome P₁-450activity as compared with control cells. In contrast, no differencein AAF mutagenicity was observed. Analysis of the AAF metaboliteprofile indicated that cells expressing the transfected cytochromeP₁-450 gene produced 8-fold more N- and 7-hydroxy-AAF than controlcells. The similarity in mutagenic responses between controlcells and cells bearing the transfected cytochrome P₁-450 genemay be due to the low deacetylase activity of AHH-1 cells. Theseobservations indicate that this vector and expression systemare suitable for introducing novel metabolic activities intothe AHH-1 cell line.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts

N-Ethyl-N-nitrosourea (ENU) forms several major adducts uponreaction with DNA, of which ethylation at the O⁶ position ofguanine and the O⁴, O² and N³ positions of thymine have beenimplicated to be mutagenic lesions. To investigate what specifickinds of ENU-induced mutations were affected by the repair abilityof O⁶-alkylguanine-DNA alkyltransferase (AGT), we examined themutations in the hypoxanthlne (guanine) phosphoribosyltransferasegene (hprt) in 87 independent mutants derived from ENU-treatedAGT proficient (Mer⁺) or deficient (Men^–) diploid humanfibroblasts. Of the characterized mutations, 97% were singlebase substitutions. The major difference in the mutation spectrawas that the frequency of G·C to A·T transitionswas significantly higher in Mer^– mutants (16/38) thanin Mer mutants (4/33). The results indicate that AGT removesO⁶-ethylguanine, thus protecting human cells from parts of thecytotoxic and mutagenic effects of ENU. A high frequency ofT· to A·T transversions induced by ENU was observedin both Mer⁺ (52%) and Mer^– (34%) mutants. This type ofmutation was less frequently observed (10%) in N-methyl N'-nitro-N-nitrosoguanidine(MNNG)-induced mutants derived from the same Mer⁺ cells in ourprevious report (J. Mol. Biol., 221, 421, 1991). Comparisonof alkylating lesions formed by MNNG and ENU indicates thatO² and N³-ethylthymine are potent mutational adducts for T toA transversions. The occurrence of ENU induced T·A basepair transverslons showed a strong strand bias; 35/37 were locatedon the non-transcribed strand, assuming thymine is the mutageniclesion. The result suggests a difference in repair capacityof ethyithymine on the two strands. In addition, this type ofmutation preferentially occurred at 5'-Pu-T sequences.     

Age dependent expression of cytochrome P-450b and metabolism of the potent carcinogen 2-nirofluorene in the rat lung

Northern and Western blot analyses, and analyses of microsomalmetabolism of the carcinogen 2-nitrofluorene (NF) were conductedwith the aim of studying age dependent cytochrome P-450_b levelsin the rat lung. The level of P-450_b homologous mRNA and correspondingprotein is very low in lungs from fetal and newborn rats. Thelevels then increase between 3 and 4 weeks of age, and reachadult levels at 6–8 weeks. No sex differences were detectedwith regard to lung P-450_b mRNA levels or catalytical activities.Lung microsomal metabolism of NF increased in parallel withthe accumulation of P-450_b homologous mRNA and microsomal cytochromeP-450_b protein concentration. Formation of the major metabolite,and potent mutagen, 9-hydroxy-2-nitrofluorene (9-OHNF) was significantlyinhibited by addition of polyclonal anti-P-450_b-IgG, and byaddition of the inhibitor proadifen to incubations with lungmicrosomal protein. It is postulated that the observed, profoundage-related differences in level and activity of lung cytochromeP-450_b are likely to affect both availability and the ratioof metabolic detoxification and activation of chemical carcinogensdeposited in the lung.