The significance of covalent binding of catechols to proteins in vivo

When rats were dosed with 14 g/kg ³H-isoproterenol, ³H-radioactivity was measurable in the liver until 48 h. This amount was not different in livers of animals which have been pretreated with diethyl maleate.After exhaustive extraction, a significant amount of ³H-radioactivity from isoproterenol could be detected in the proteins of total liver (homogenate), of cytosol and of microsomes. In the cytosol fraction of diethyl maleate pretreated animals twice the amount of isoproterenol-radioactivity was found in the extracted proteins compared to controls. In the microsomal fraction there was no difference between diethyl maleate pretreated and control animals in the amount of radioactivity incorporated into proteins.In all fractions the radioactivity measurable in the extracted proteins declined with a half life time of about 24 h.The in vivo results on covalent binding of isoproterenol are compared to the irreversible protein binding of ethinyloestradiol in vivo. Quantitatively, these in vivo data are compared to the results on irreversible protein binding obtained during incubations of isoproterenol or ethinyloestradiol with rat liver microsomes.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

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 = μanol 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 DNAbinding 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.     

In vitro covalent binding of the pyrethroids cismethrin,cypermethrin and deltamethrin to rat liver homogenate and microsomes

Phenobarbital-induced rat liver homogenate and microsomes were used to study covalent binding of l4C-labelled (at the alcohol moiety) cismethrin, ¹⁴C-labelled (at the alcohol and acid moieties) cypermethrin, and ¹⁴C-labelled (at the alcohol and acid moieties) deltamethrin. Covalent binding was dependent on pyrethroid concentration. With liver homogenate, inhibition of esterases by tetraethylpyrophosphate and of mitochondrial respiration by rotenone or potassium cyanide only slightly altered the covalent binding level. With microsomes, inhibition of cytochrome P-450 and mixed function oxidases by carbon monoxide and piperonyl butoxide reduced the covalent binding so far as to be nearly absent. Eighty percent inhibition of epoxide hydrolase decreased the covalent binding by 50%. The comparison of data between alcohol and acid labelling of the same pyrethroid suggested that, in vitro, the whole molecule is bound to proteins and that hydrolysis can occur afterwards. The experiments stress the role of cytochrome P-450-dependent monoxygenases in the covalent binding process.     

Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat

1,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-term bioassay using gavage in corn oil (24 and 48 mg/kg/day), but not by inhalation (up to 150–250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Female F-344 rats (183–188 g) were exposed to [1,2-¹⁴C]- DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l=80 ppm for 4 h) or to a peak concentration (up to 18 mg/l=4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/kg. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymatically hydrolyzed to the 3-nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density, indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The level of DNA adducts was expressed in the dose-normalized units of the Covalent Binding Index, CBI = (mol adduct per mol DNA nucleotide/mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for constant-low and peak DCE exposure levels. In the lung, the respective values were 0.9 and 31. It is concluded that the DNA damage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-level inhalation exposure.     

Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo

It was the aim of this investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or without pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [¹⁴C]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The limit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [¹⁴C]- and [³H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside or base adducts are expected. The respective limits of detection were at 0.07 and 0.04 CBI units for the ¹⁴C and ³H labels, respectively. The experiments with [¹⁴C]- and [³H]DEHA, labeled in the alcohol moiety and administered to mice, revealed a minute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which had been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for ¹⁴C and 0.01 to 0.12 for ³H resulted. Determination of the level of ¹⁴CO₂ expiration revealed a linear correlation with the specific activity of DNA. Experiments with 2-ethyl[1-¹⁴C]hexanol performed with both rats and mice allowed the conclusion that most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals with unlabeled compound had no effect on the DNA radioactivities in either species. The present negative data, in conjunction with other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.     

Enzymic control of irreversible binding of metabolically activated benzo(a)pyrene in perfused rat liver by monooxygenase activity

Addition of [³H]-benzo(a)pyrene to the perfusion medium of isolated rat livers results in irreversible binding of radioactivity to DNA, RNA and protein. Binding to DNA accounted for about 0.1% of the total radioactivity which was bound in livers from animals treated with oil or saline and was increased by a factor of 3–5 after pretreatment of the animals with -naphthoflavone or with phenobarbital. When the inhibitiors of monooygenase activity, -naphthoflavone or metyrapone, were present in the perfusion medium, irreversible binding was reduced in livers from both -naphthoflavone- and phenobarbital-pretreated animals, irrespective of the inhibitor used.In livers from animals treated with oil or saline protein and a RNA fraction containing tightly associated protein were able to bind [³H]-benzo(a)pyrene metabolites to about the same extent but after induction by pretreatment with -naphthoflavone binding to the RNA fraction was enhanced to a much higher extent than binding to the protein fraction. Pretreatment with phenobarbital did not result in an increased irreversible binding to RNA and protein.A considerable amount of 15–25% of the total radioactivity added to the perfusion medium was excreted into the bile after treatment of the animals with the tested inducers of monooxygenase activity compared to an excretion of 3% in animals treated with oil or saline.The results indicate that nucleic acid and protein adduct formation in the liver is controlled by the action of the cytochrome P-450-dependent monooxygenases.In part subject of the doctoral thesis of Erik Klaus, Fachbereich Biologie, University of Mainz     

Trenbolone growth promotant: covalent DNA binding in rat liver and inSalmonella typhimurium,and mutagenicity in the Ames test

DNA binding in vivo: [6,7-³H]-trenbolone (-TBOH) was administered p.o. and i.p. to rats. After 8 or 16 h, DNA was isolated from the livers and purified to constant specific radioactivity. Enzymatic digestion to deoxyribonucleotides and separation by HPLC revealed about 90% of the DNA radioactivity eluting in the form of possible TBOH-nucleotide adducts. The extent of this genotoxicity, expressed in units of the Covalent Binding Index, CBI = (mol TBOH bound per mol nucleotide)/(mmol TBOH administered per kg body weight) spanned from 8 to 17, i.e. was in the range found with weak genotoxic carcinogens.Ames test: low doses of -TBOH increased the number of revertants inSalmonella strain TA100 reproducibly and in a dose-dependent manner. The mutagenic potency was 0.2 revertants per nmol after preincubation of the bacteria (20 min at 37° C) with doses between 30 and 60 g per plate (47 and 94 g/ml preincubation mixture). Above this dose, the number of revertants decreased to control values, accompanied by a reduction in survival. The addition of rat liver S9 inhibited the mutagenicity.DNA binding in vitro: calf thymus DNA was incubated with tritiated -TBOH with and without rat liver S9. Highest DNA radioactivities were determined in theabsence of the activation system. Addition of inactive S9 (without cofactors) reduced the DNA binding by a factor of up to 20. Intermediate results were found with active S9.DNA binding in Salmonella: -TBOH was irreversibly bound to DNA isolated fromS. typhimurium TA100 after incubation of bacteria with [³H]-TBOH.Conclusions: Covalent DNA binding appears to be the mechanism of an activation-independent (direct) mutagenicity of TBOH which is not easily detected because of the bactericidal activity. The genotoxicity risk arising from exposure of humans to trenbolone residues in meat was estimated using the in vivo data and compared to that from the exposure to unavoidable genotoxins aflatoxin B₁ and dimethylnitrosamine. It is concluded that trenbolone residues represent only a low genotoxic risk.Part of this study was reported at the 18th Annual Meeting of the Union of the Swiss Societies of Experimental Biology, Basel, March 20–21, 1986. The abstract appeared in Experientia 42, 697 (1986)     

Reactions of vinyl chloride with RNA and DNA of various mouse tissues in vivo

The irreversible binding of ¹⁴C-vinyl chloride metabolites to RNA and DNA of mouse brain, lung, liver, kidney, spleen, pancreas, and testes after a single i.p. injection has been studied. Hydrolysates of nucleic acids from selected organs were separated on Aminex A6 for quantitation of alkylation products.Radioactivity in nucleic acids was registered in all of the studied organs with the exception of brain. RNA from spleen, pancreas and liver, and DNA from spleen and liver contained the highest amounts of radioactivity. In nucleic acids from spleen and pancreas, both organs of high metabolic activity, the entire radioactivity was found metabolically incorporated as C₁-fragments. In RNA of kidney and liver, a large part of the radioactivity was also present as incorporated C₁-fragments, but 3,N⁴-ethenocytidine (in kidney) as well as 1,N⁶-ethenoadenosine and 1,N⁶-ethenoadenine (in kidney and liver) were identified as alkylation products. In liver DNA, incorporation of C₁-fragments was insignificant, indicating different interactions of vinyl chloride metabolites (C₁-fragments and alkylating products) with RNA and DNA. The elution profiles of radioactivity in hydrolysates of liver DNA were dominated by an alkylation product of unknown structure (probably a derivative of deoxyguanosine). Possibly, 1,N⁶-ethenodeoxyadenosine and 1,N⁶-ethenoadenine were also present in liver DNA.The results are consistent with the ability of vinyl chloride to act as a multipotent carcinogen by alkylation of DNA in several tissues.     

Irreversible binding of 3-14C-antipyrine to hepatic protein in vivo and in metabolizing liver microsomes

Summary After i.p. injection of 3-¹⁴C-antipyrine (10 mole=1.9 mg with 10 Ci per 10 g of body weight) to mice radioactivity was irreversibly bound to liver proteins. The irreversible binding reached maximal values of 0.15 nmole/mg protein in liver microsomes after 30–60 min.During 60 min incubation with liver microsomes of mice and rabbits (phenobarbital pretreated) and a NADPH-regenerating system 3-¹⁴C-antipyrine was irreversibly bound to microsomal protein at a rate of 1.5 nmole/mg protein (mouse) and 3 nmole/mg protein (rabbit).In identical incubates with rabbit liver microsomes the 4-hydroxylation of antipyrine was 24 nmole/mg protein in 60 min and formaldehyde production from antipyrine 3 nmole/mg protein in 60 min.In incubates with rabbit liver microsomes the binding rate was 80–90% inhibited by 1mM metyrapone, SKF 525-A and trichloropropene epoxide respectively; 4-hydroxylation was 70–80% inhibited by the same substances. In the presence of 1 mM GSH, cysteine or ethylene diamine binding was 30–40% inhibited, whereas 4-hydroxylation showed no inhibition.Some of the results were presented at the 17th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, March 1976     

Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

1. ¹⁴C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h: 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein.
2. The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extractions removed 60–70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital.

     

Forskolin blocks carbachol-mediated ion-permeability of chick myotube nicotinic receptors and inhibits binding of 3H-phencyclidine to Torpedo microsac nicotinic receptors

Summary Forskolin, a commonly used adenylate cyclase activator, was found to inhibit reversibly the carbachol-induced ion-translocating capacity of the nicotinic acetylcholine receptor (nAChR) on chick myotubes in a dose- (IC₅₀ = 20 M) and time-dependent manner. This effect was not correlated to increases in cellular cAMP. Forskolin, at a concentration (50 M) that totally blocked the carbachol-induced ⁸⁶Rb influx, caused no change in carbachol or -bungarotoxin binding to chick myotube nAChR in situ. In contrast, in the presence of carbachol, forskolin inhibited (IC₅₀ = 10 M) the binding of ³H-phencyclidine, a putative nAChR ion-channel ligand, to Torpedo microsac nAChR. Inhibition of ³H-phencyclidine binding in the absence of carbachol was not complete. Membrane leakage studies on myotubes, measuring ³H-efflux from 2-deoxy-d(1-³H)-glucose loaded cells and electrophysiological measurements of membrane properties supported the interpretation that forskolin induced decreases in plasma membrane permeability. In conclusion, forskolin blocks the carbachol-mediated increase in permeability of the nAChR channel by (1) binding to the ion-channel (open state) and (2) generally perturbing the plasma membrane function possibly by interfering with the protein-lipid interface. Send offprint requests to Johan Haggblad     

The fate of phenylhydroxylamine in human red cells

Summary Phenylhydroxylamine added to human red cells under aerobic conditions and in the presence of glucose was partly reduced to aniline. About half the hydroxylamine was recovered as amine after a 2-hr incubation. The aniline, after acetylation, was identified as acetanilide by melting point, R_f-value in TLC as well as UV, IR, and NMR spectroscopy.The fate of the remaining phenylhydroxylamine was followed by use of ¹⁴C-labeled phenylhydroxylamine. About 30% of the total radioactivity was bound to hemoglobin or other proteins and about 20% was found in highly polar low-molecular substances which were insoluble in organic solvents.The elucidation of the sites at which phenylhydroxylamine was bound to hemoglobin was complicated by the lability of the bonds. When purified human hemoglobin had reacted with radioactive phenylhydroxylamine, large proportions of the radioactivity bound to hemoglobin were removed by treatment with acid or with PMB for separation of - and -chains. The radioactive compound liberated from hemoglobin by acid was found to be aniline.After reaction with phenylhydroxylamine the number of SH groups titrable with PMB was found to be diminished. Pretreatment of hemoglobin with N-ethylmaleimide or PMB decreased the amount of phenylhydroxylamine bound to hemoglobin but did not fully prevent the reaction.Tryptic digestion of hemoglobin after reaction with radioactive phenylhydroxylamine yielded tryptic peptides with lower specific activity than that of hemoglobin. Chymotryptic digestion of the tryptic core yielded a core with specific activity much higher than that of hemoglobin. Fingerprinting of the tryptic or chymotryptic hydrolyzates showed the presence of peptides with high and other ones with low or no radioactivity and of radioactive compounds which did not react with ninhydrin.In the covalent binding of phenylhydroxylamine to globin the SH group 93 plays an important role, but other yet unknown sites are also reactive.     

Effects of vitamin A on the metabolism and mutagenicity of 2AAF in vitro and on the covalent binding to rat liver DNA/RNA in vivo

Vitamin A has been shown to affect the in vitro metabolism of 2AAF. At low concentrations of retinol or retinyl palmitate, a decreased production of ring-hydroxylated as well as deacetylated and TV-hydroxylated metabolites was observed, measured by high performance liquid chromatography. The increased mutagenicity of 2AAF observed after addition of vitamin A in the Ames test cannot therefore be explained as a result of stimulated N-hydroxylation. However, the addition of retinol was found to enhance the mutagenicity of the metabolite N-OH-2AAF in the presence of an S-9 fraction of rat liver homogenate. No differences with regard to the covalent binding of 2AAF or its metabolites to rat liver DNA/RNA in vivo could be demonstrated in animals fed diets with normal or high vitamin A content.Abbreviations Used 2AF 2-aminofluorene - 2AAF 2-acetylaminofluorene - GLU-P-1 2-amino-6-methyl-dipyrido(1,2-a3,2-d)imidazol - GLU-P-2 2-amino-dipyrido(1,2-a3,2-d)imidazol - N-OH-2AAF N-hydroxy-2-acetylaminofluorene - OAAT orthoaminoazotoluene - TRP-P-1 3-amino-3,4-dimethyl-5H-pyrido(4,3-b)indole - TRP-P-2 3-amino-1-methyl-5H-pyrido(4,3-b) indole     

Reversible metabolism of vitamin K-vitamin K epoxide: Modeling considerations and limitations

Due to the cyclical natural of the vitamin K-vitamin K epoxide system, a two-compartment reversible metabolism model was used to describe this interconversion. In attempting to apply this model to the vitamin K-vitamin K epoxide cycle using literature data from dogs, interconversion and elimination clearances were obtained which are not physiologic. Consequently, the assumptions of the model were reexamined with respect to their validity. One critical assumption of the twocompartment model for interconversion is that it can only be applied in the absence of flow limitations. To determine what effect flow limitations may exert on the vitamin K and vitamin K epoxide apparent blood clearances, a model separating the liver from the blood compartment was proposed assuming the interconversion and metabolism of vitamin K and its epoxide occurred only within the liver. Simulated data suggested that if the reversible metabolic clearance values exceeded the distributional clearance terms, all the apparent clearances calculated using blood concentration-time data were in error. It is suggested that a two-compartment interconversion model might be too simplistic for the vitamin K-vitamin K epoxide cycle where the reversible metabolism is efficient and the distributional clearance may be rate limiting.Glossary of symbols Dose ^E Dose of vitamin K epoxide - Dose ^K Dose of vitamin K AUCs calculated from blood concentration-time profile AUC _E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _E ^K Area under curve for vitamin K after epoxide dose - AUC _K ^K Area under curve for vitamin K after vitamin K dose - AUC _E ^K Area under curve for vitamin K epoxide after vitamin K dose AUCs calculated from liver concentration-time profile AUC _L,E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _L,K ^E Area under curve for vitamin K after epoxide dose - AUC _L,K ^K Area under curve for vitamin K after vitamin K dose - AUC _L,E ^K Area under curve for vitamin K epoxide after vitamin K dose Clearances calculated from blood AUCs Cl _E ^B Elimination clearance of vitamin K epoxide - Cl _K ^B Elimination clearance of vitamin K - Cl _EK ^B Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^B Conversion clearance from vitamin K to vitamin K epoxide Clearances calculated from liver AUCs Cl _E ^L Elimination clearance of vitamin K epoxide - Cl _K ^L Elimination clearance of vitamin K - Cl _EK ^L Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^L Conversion clearance from vitamin K to vitamin K epoxide Distributional clearances Cl _K ^BL Clearance of vitamin K from the blood to the liver - Cl _K ^LB Clearance of vitamin K from the liver to the blood - Cl _E ^BL Clearance of vitamin K epoxide from the blood to the liver - Cl _E ^LB Clearance of vitamin K epoxide from the liver to the blood Supported by a grant from The American Heart Association, Kentucky Affiliate.     

Synthesesteigerung und Abbauhemmung bei der Vermehrung der mikrosomalen Cytochrome P-450 und b-5 durch Phenobarbital

Summary Heme moieties of microsomal rat liver cytochromes P-450 and b-5 were labeled with.¹⁴C-Aminolevulini cacid. The half life of the b-5 heme radioactivity was found to be 45 hrs, that of the P-450 heme radioactivity was 22 hrs.Treatment of fed rats with Phenobarbital (80 mg/kg i.p. and 1 PB in the drinking water) for 48 hrs increased the concentration of cytochrome P-450 up to 200%, only by induced synthesis. In starved rats treated with Phenobarbital, P-450 concentration was increased up to 400%, by both induced synthesis and inhibition of breakdown.Microsomal P-450 cytochrome was determined in rat liver homogenate and in suspensions of rat liver microsomes. The amount of P-450 obtained in the isolated microsomal fraction was compared with the P-450 content in the liver homogenate. Since P-450 is a microsomal hemoprotein this relation can be correlated with the microsomal protein, in order to to calculate the real content of microsomal protein in the liver homogenate. It was found to be 65 mg/g of liver, demonstrating, that 31% of the 209 mg of total protein/g of liver consists if microsomal protein.

     

The metabolism and binding of ( 14 C)mycophenolic acid in the rat

Seventeen h after the intraperitoneal administration of 33 mg/kg of [¹⁴C]mycophenolic acid to rats, radioactivity was bound to the tissues of the intestines, bladder, stomach, kidney, liver and lung in decreasing order; no binding to spleen tissue was observed. In vitro incubations of the agent with macromolecules resulted in the binding of radioactivity to salmon sperm DNA and to bovine plasma albumin, the extent of binding being increased and decreased, respectively, in the presence of a rat liver microsomal system. The binding was apparently covalent since repeated purification procedures failed to release the bound radioactivity; heating of [¹⁴C]mycophenolic acid bound-DNA in n hydrochloric acid at 100° for 2 h caused the release of the bound radioactivity. Under the conditions described, 43% of the administered radioactivity was excreted in the urine (33%) and faeces (10%); the urine contained free mycophenolic acid (13%), mycophenolic acid glucosiduronate (17%) and an uncharacterized metabolite (3%).     

DNA binding,adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal,Kupffer and endothelial cells

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B₁, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP⁺ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B₁ to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B₁ had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B₁ in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B₁. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B₁ to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B₁, a dose-dependent aflatoxin B₁ binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxin B₁.This project was supported by the Deutsche Forschungsgemeinschaft (SFB 302). B. Schlemper was the recipient of a European Science Foundation Scholarship     

Lack of correlation between formation of reactive metabolites and thymic atrophy caused by 3, 4, 3′, 4′-tetrachlorobiphenyl in C57BL/6N mice

The possible role of active metabolites of 3, 4, 3, 4-tetrachlorobiphenyl (TCB) in causing thymic atrophy was investigated using inbred strains of mice. The generation of reactive species which bind covalently to cellular proteins was used to monitor the formation of active TCB metabolites. The amount of in vitro covalent binding of TCB to proteins by liver microsomes was increased markedly by pretreatment of AHH-responsive C57BL/6N mice with either 3-methylcholanthrene (MC) or TCB itself, although these two inducers were not effective in AHH-nonresponsive DBA/2N mice. MC treatment also caused an induction of microsomal TCB-binding activity in all of the (C57BL/6N x DBA/2N) F₁ mice. Moreover, among 38 individuals of [(C57BL/6N) (DBA/2N) F₁ x DBA/2N] backcross, 23 mice responded to MC with respect to microsomal TCB-binding activity while others did not. These results suggest that the conversion of TCB to protein-bound metabolites is mediated by particular form(s) of cytochrome P-450 which is (are) induced by an Ah receptor mechanism. In order to ascertain wheter the active TCB metabolites play a role in causing thymic atrophy, ¹⁴C-labeled TCB was administered IP to C57BL/6N mice and the amount of covalent binding of radioactive metabolites to tissue proteins was determined. The in vivo binding was evident in the liver, particularly in the microsomal fraction, on the basis of protein content. In contrast, the thymic proteins contained no measurable amounts of bound radioactivity even when the mice showed marked thymic atrophy. These data suggest that thymic atrophy caused by TCB is not likely to result from the generation of reactive metabolites.Abbreviations Used TCB 3, 4, 3, 4-tetrachlorobiphenyl - PCB polychlorinated biphenyl(s) - TCDD 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin - MC 3-methylcholanthrene - PB phenobarbital - AHH aryl hydrocarbon hydroxylase     

Synthesis,antitumor activity evaluation,and DNA‐binding study of coumarin‐based agents

A novel series of coumarin‐thiadiazole heterocycle derivatives was synthesized by the nucleophilic substitution reaction. The synthesized compounds were structurally verified by IR, ¹H NMR, ¹³C NMR, mass spectra, and elemental analyses. The antitumor activity of the synthesized compounds was evaluated through DNA binding assays and the 60‐cell line panel according to the US NCI‐DTP protocol or a selection of human tumor cell lines: breast cancer (MCF‐7), liver cancer (HepG‐2), and colorectal cancer (HCT‐116). Most of the compounds had better DNA/ethidium bromide fluorescence quenching rather than methyl green displacement, suggesting superior DNA intercalation over DNA groove binding. Compounds 8 and 14b showed the best quenching effect with K_SV = 4.27 × 10⁵ M^?1. Moreover, the results for compounds 8 , 4c , and 4e revealed a possible dual DNA binding mode with the intercalation to be superior, with K_SV 4.27 × 10⁵, 3.96 × 10⁵, and 3.51 × 10⁵ M^?1, respectively, compared to 42%, 45%, and 43% methyl green displacement, respectively. Out of the 60‐cell line panel, the leukemia HL‐60 cell line was the most susceptible to growth inhibition when treated with 14a , resulting in 61% growth, followed by the lung carcinoma cell line NCI‐H522 showing 67% growth when treated with 9 . Moreover, compound 10c had an IC₅₀ value of 24.9 μg/mL against the HepG‐2 cell line.

     

Tests for mutagenicity in salmonella and covalent binding to DNA and protein in the rat of the riot control agent o-chlorobenzylidene malononitrile (CS)

The aim of this study was to determine whether o-chlorobenzylidene malononitrile (CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [¹⁴C]-label at the benzylic carbon atom. It was administered i.p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liver and kidney DNA was isolated after 8,25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 10⁵ times lower than that of the strong hepatocarcinogen aflatoxin B₁, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. CS was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose levels used, 1,000 and 2,000 g CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 g per plate, and a subsequent fall below control values at 1,000 g. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of CS so that the calculated mutation frequencies, i.e., the number of revertants per number of surviving bacteria, increased with doses up to 500 g. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.