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Measurement of reaction products of alkylating agents with proteins has been used as a monitor of in-vivo exposure to over 30 such compounds. Doses in animals exposed to directly-acting alkylating agents (e.g., methyl methanesulfonate, ethylene oxide) are directly related to the production of alkylated amino acids in haemoglobin. The erythrocyte dose of alkylating agent, calculated from the extent of haemoglobin alkylation, is in some cases related to liver and extra-hepatic DNA doses; thus, detection of alkylation of haemoglobin may be taken as an indication of a reaction at the carcinogenic target site.

Cell toxicity by BZQ could not be explained by free radical formation and thus further work has been undertaken to elucidate a possible mechanism of cell killing. By using radiolabelled BZQ, in vitro DNA-drug binding has been investigated. The effect of salt, buffer and drug concentrations was determined in the pH range 4.0 to 8.0. The influence of in situ oxidation and reduction on BZQ binding was also studied as a function of pH. In an effort to ascertain any base specificity of BZQ binding the homopolymers, Poly[dG]. Poly[dC] and Poly[dA]. Poly[dT] were treated with radiolabelled BZQ in the pH range 4.0 to 8.0. A fluorescence assay was used to demonstrate the possible involvement of DNA cross-linking in cellular activity. From this work, it was concluded that BZQ functions as a bifunctional alkylating agent by an acid-assisted aziridine ring-opening mechanism and that other factors including oxidation or reduction are much less important.

The interaction of 4-(N-chloroethyl-N-methylamino)-benzyl-gamma-amide ATP (I) and the corresponding beta-amide of ADP (II) with rat liver acetyl-CoA carboxylase was studied. Both analogs were shown to cause affinity modification of the enzyme. ATP and GoAS Ac protected the enzyme against inactivation. HCO3- increased the rate of carboxylase inactivation by analogs I and II (2.5- and 1.5-fold, respectively). The alkylating amides did not influence the rate of the bicarbonate-dependent [14C]-ADP-ATP exchange and inhibited the enzyme-catalyzed reaction of [14C]-CoAs Ac----CoAS Mal exchange, which testifies to the localization of the modified group in the CoAS Ac-binding site of the enzyme active center. Based on the affinity modification and analog size, it was found that the distance between the ATP- and CoAS Ac-binding sites of the enzyme active center can vary from 0.8 to 1.2 nm.

Single-stranded primed M13mp2 templates and double-stranded templates were treated with either dimethyl sulfate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine and used for DNA synthesis in vitro. Methylation inhibits the ability of the molecules to serve as templates. When either E. coli DNA polymerase I or AMV reverse transcriptase were used as polymerases, DNA synthesis terminated one nucleotide 3' to the site of adenine residues in the template. Heating of the templates resulted in the appearance of additional termination bands one nucleotide before the site of G's in the template. We assume that methylated A's but not methylated G's are blocks to in vitro DNA synthesis and that heating converts a portion of the sites of methylated G to AP sites which are blocks to synthesis.

The synthetic vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) has been shown to influence a wide range of cell-membrane-related events. Accordingly, the effect of dDAVP on membrane transport of various alkylating agents and amino acids was evaluated in L5178Y lymphoblasts in vitro. dDAVP stimulated melphalan uptake but conversely inhibited uptake of nitrogen mustard, choline (the natural transport substrate for the nitrogen mustard carrier), and leucine. No effect on the uptake of cyclophosphamide or glutamine was observed. Increased melphalan uptake was due to effects on both substrate influx and efflux. The effect of dDAVP on melphalan influx was particularly complex: dDAVP stimulated melphalan influx by amino acid transport system ASC but inhibited influx by system L, resulting in a net increase in unidirectional drug influx. Melphalan efflux was inhibited by dDAVP. Decreased uptake of nitrogen mustard, choline and leucine was due, at least in part, to decreased substrate influx. However, the mechanisms of inhibition were dissimilar: inhibition of substrate influx was non-competitive for choline but competitive for leucine. In conclusion, dDAVP induced diverse but apparently specific effects on membrane transport of several alkylating agents and amino acids. Since the accumulation of alkylating agents such as melphalan within tumor cells is a major determinant of cytotoxicity, dDAVP may have a role as a biological response modifier.

A method is presented for the characterization of the reaction order and rate constant for chemically and metabolically generated reactive electrophilic intermediates. The procedure employs flow kinetics and trapping of the electrophilic species. Reactive agents or metabolic intermediates are passed through a column containing a bound nucleophilic reagent. The electrophilic species reacts at a characteristic rate while moving through the column at a fixed pH, temperature and rate of flow. The alkylation products formed during this reaction are quantified in individual column segments which correspond to time intervals. This provides data for the calculation of the lifetime of the short-lived species. The method may be used for agents having half-lives of 1 s to 30 min. Metabolic intermediates need not be isolated. Data is presented for the reaction rates of 1-methyl-1-nitrosourea (MNU), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and iodomethane. A metabolic activation system is described for the conversion of acetoxy-methylmethylnitrosamine (DMN-OAc) to hydroxymethylmethylnitrosamine (DMN-OH) and measurement of the stability of that intermediate. Hydroxymethylmethylnitrosamine was found to have a half-life of 28s at pH 7.4, 37 degrees C.

DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Both, in E. coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems. Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse. The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar. These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

Adult male South African clawed frogs, Xenopus laevis, were mutagenized by 3-day immersion in aqueous solutions of ethyl methanesulfonate (EMS), diethyl nitrosamine (DEN), or ethyl nitrosourea (ENU), or by acute exposure to gamma radiation. They were then spawned repeatedly at 2-week intervals with untreated females, and embryonic survival of the progeny was used to assess genetic damage. Recessive lethal effects were assessed from reduced survival of androgenetic haploid progeny. Neither recessive nor dominant lethal effects were obtained after exposure to 100 mg/liter EMS or 2 g/liter DEN. At 250 mg/liter EMS, peak dominant lethality occurred 3-5 wk after treatment. Most embryos hatched, but many were abnormal and died shortly after hatching. Haploid survival was significantly reduced over a broader period, from 1 to 13 wk after mutagenesis. Treatment with 75 mg/liter ENU produced effects similar to the 250-mg/liter EMS mutagenesis. At 400 mg/liter EMS, the frequency and severity of the effects on both diploid and haploid embryos were increased over the lower dose. Gamma irradiation at 1,500 R produced effects similar to the 400-mg/liter mutagenesis, except that peak dominant lethality extended from 1 to 7 wk.

Several recent and major advances in the technology of mass spectrometry (MS) have greatly promoted use of this technique for the study of the interaction of alkylating agents with biomolecules. MS, in combination with gas chromatography (GC), may now be used to quantify adducts of carcinogens with proteins at levels down to 20 pmol/g protein. Soft ionization techniques have proved invaluable in determining the structure of carcinogen adducts with both DNA and proteins, and the newly developed tandem MS promises to be of considerable use in the characterization of complex carcinogen adduct mixtures.

The relationship in Drosophila males between chemical reaction pattern of mono-functional alkylating agents (AA), described in terms of primary alkylation pattern with DNA and proteins as well as the Swain--Scott s factor, and their biological effectiveness were investigated. The agents chosen for comparative analysis were the nitrosamides ENU and MNU, the methanesulfonic esters iPMS, EMS and MMS, the dialkylsulfate DMS, and the nitrosamines DEN and DMN. Parameters of their biological activity were mortality (LC50) of treated adult males, induction in post-meiotic stages of X-chromosomal recessive lethal mutations and 2--3 translocations after either adult feeding or injection. Induced frequencies of recessive lethals, determined for each AA with a range of concentrations, served as biological dosimeter for interaction with target DNA in the germ line. The results are interpreted as indicating for these AA a causal connection between the pattern of primary alkylation of DNA and the quality of genetic damage observed. 1. The agent with the lowest s value, ENU, and its pendant DEN, failed to produce translocations at mutation frequencies that reached 44% for ENU. The highest chromosome-breaking activity was attributed to AA with high s, MMS and DMS. For MMS, the proportions of translocations (T) to mutations (M) approximately reached a 1 : 1 ratio in stored spermatozoa, at a recessive-lethal frequency of 14%. Ability to break chromosomes, as indicated by the T : M ratios, decreased in the sequence MMS greater than or equal to DMS, MNU greater than DMN greater than EMS greater than iPMS greater than ENU = DEN. 2. Nearly the reversed sequence in relative mutagenci effectivenss was obtained when the (directly acting) AA were arranged on the basis of their CM4/LC50 ratios (CM4, the exposure condition producing 4% recessive lethals after injection): ENU greater than EMS greater than iPMS, MNU greater than MMS = DMS. 3. Among the AA, EMS had a somewhat unique position, in that it was slightly less effective in the translocation test, and also less cytotoxic but more mutagenic in the recessive-lethal test than one would expect from its s value. This is taken as an indication of the influence on biological effectiveness of factors other than the s value, e.g. methylation versus ethylation and the lipid/water partition ratio. An example of the latter was also provided by DMS which, although having the same s as MMS, with its 5-fold higher lipid/water partition ratio, was more toxic than MMS. 4. For those AA that were clearly active in the translocation tests--MMS, DMS, MNU, DMN and EMS--delayed formation of exchanges was observed. Only in 17 out of 555 translocation tests with positive response translocations were already found in progeny from unstored spermatozoa. Consequently, it was concluded that performance of storage experiments in Drosophila is an absolute necessity for the detection of this type of rearrangement by AA. 5...

Seven classical alkylators were tested for activity against the continuous human medulloblastoma cell line TE-671 grown in vitro and as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide (4-hydroperoxycyclophosphamide in vitro), iphosphamide (4-hydroperoxyiphosphamide in vitro), phenylketocyclo-phosphamide, phenylketoiphosphamide, Asta Z 7557, and thiotriethyl-enephosphoramide. All agents were active, with melphalan demonstrating the most activity in vitro and in vivo. Comparative studies of cyclophosphamide and phenylketocyclophosphamide revealed partition coefficients (log P) of 0.73 and greater than 1.69, respectively, and cyclophosphamide exhibited greater cytotoxic activity in post- (equitoxic) drug administration murine plasma. Hematological toxicity was limited to leukopenia/neutropenia for both of these agents. These studies suggest that the classical alkylators may have a role in the treatment of medulloblastoma and provide a means to further analyze their therapeutic potential.

Rat liver microsomes catalyzed the biotransformation of the clinically important nitrosourea anticancer agents 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to alkylating metabolites that bound covalently to microsomal protein and to DNA. The enzyme-mediated microsomal alkylation required NADPH and oxygen and was inhibited by carbon monoxide, indicating the participation of a cytochrome P-450-dependent monooxygenase. Additional studies with inhibitors such as piperonyl butoxide and with the inducers 3-methylcholanthrene and phenobarbital were consistent with this view. In contrast to these observations on the formation of alkylating metabolites, carbamylation reactions were not affected significantly by microsomal metabolism. Reduced glutathione, cysteine or N-acetylcysteine decreased the microsomal alkylation by MeCCNU and produced a corresponding increase in the formation of polar metabolites that was resolved by HPLC as three distinct N-acetylcysteine-MeCCNU adducts. The addition of semicarbazide to the reaction decreased microsomal alkylation by 30%, indicating that the formation of the alkylating species may proceed via an aldehyde intermediate. Renal microsomes were not found to catalyze the alkylation reaction. Moreover, MeCCNU inhibited the renal slice accumulation of p-aminohippuric acid only in the presence of liver microsomes and NADPH, suggesting that a liver metabolite may be responsible for the renal toxicity of the parent nitrosourea.

Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.

The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract.

Although formation of DNA adducts has been postulated for several halomethanes, no chemical identification of such adducts has been performed so far. There is, however, evidence that methyl chloride does not act biologically as a DNA methylating agent. 1,2-Dichloroethane and 1,2-dibromoethane are activated through conjugation with glutathione. There is some evidence for formation on an N-7 adduct of guanine which carries an ethyl-S-cysteinyl moiety. Extensive work has been published on adducts of vinyl chloride, both in vitro and in vivo. The major DNA adduct is 7-(2-oxoethyl)guanine; a minor adduct appears to be N2,3-ethenoguanine. Other "etheno" adducts, i.e., 1,N6-ethenoadenine and 3,N4-ethenocytosine, are readily formed with DNA, vinyl chloride, and a metabolizing system in vitro and with RNA in vivo, but are usually not detected as DNA adducts in vivo. The data on DNA alkylation by vinyl chloride (and vinyl bromide) metabolites are compared with those of structurally related compounds (acrylonitrile, vinyl acetate, vinyl carbamate).

In Drosophila the connection between primary alkylation pattern and genetic activity of 8 mono-functional alkylating agents (AA)--ENU, DEN, iPMS, MNU, DMN, EMS, MMS and DMS--was investigated in spermatozoa and spermatids of treated R 1 (2), y B/BS Y y+ males. The induction of ring-X loss, Y-chromosome loss, and Y-rearrangements served as parameters of their effectiveness to produce chromosomal aberrations. (1) The ability to cause chromosomal losses decreased in the sequence MMS = DMS greater than MNU = DMN greater than EMS greater than iPMS greater than ENU = DEN. This ranking is consistent with the sequence in effectiveness in producing translocations. (2) The low frequencies of Y-chromosome rearrangements made any meaningful comparison between the AA impossible. The diagnostic value of such tests is poor, because even at toxic concentrations large numbers of offspring must be scored to obtain statistically significant effects. (3) With MNU, DMN, EMS, MMS and DMS, a time-dependent increase in the frequencies of ring-X losses ("storage effect") was found. (4) A stage-by-stage comparison revealed a differential response of spermatozoa and spermatids to these AA. Mature spermatozoa tended to be more resistant to the induction of breaks by MNU, DMN, EMS and MMS. (5) A general observation with all the AA was a reduced rate in survival of X-bearing sperm after treatment with mutagen. There was, however, no apparent quantitative relationship between the shift in the sex ratios observed and the yield of chromosomal aberrations. It is concluded that differences between these AA in their selectivity to numerous nucleophiles of DNA, as expressed by their s values, account for most of the diversity in their genetic effectiveness. The significance for genetic activity of other parameters than the s value appeared from the lower activity of EMS relative to MNU, indicating that methylation was more effective than ethylation in breaking chromosomes.

Two possible mechanisms for the reported carcinogenic synergism between ethylene dibromide (EDB) and disulfiram have been investigated in vivo and in vitro, the first involving increased production of an EDB-derived glutathione mustard and the second increased production of bromoacetaldehyde. Consistent with both of these suggested mechanisms, repeated administrations of disulfiram to rats inreased liver glutathione-S-transferase activity and decreased liver low Km aldehyde dehydrogenase activity. However, when added to a rat liver S-9 fraction in vitro, disulfiram decreased transferase activity and only depressed the dehydrogenase activity after a period of preincubation. Although the mutagenic potency of EDB to Salmonella typhimurium was slightly enhanced in vitro by the addition of a rat liver S-9 fraction, the further addition of disulfiram to the assay medium produced no additional change. Similarly, the addition of a range of S-9 and S-0.5 liver fractions derived from disulfiram-treated rats also failed to enhance significantly its mutagenic potency over the normal S-9 fraction. The general implications of these findings are discussed.

Incubations with goat lung and liver microsomes were conducted to trap with exogenous glutathione (GSH) the electrophilic intermediate produced via cytochrome P-450-dependent metabolic activation of 3-methylindole (3MI). Microsomal incubation mixtures with [14C]3MI, a NADPH-generating system, and [3H]GSH produced a dual-labeled adduct which was isolated by reverse-phase high-performance liquid chromatography. Reactive 3MI intermediates were also trapped with cysteine. Adduct formation increased in proportion to the concentration of either thiol. Covalent binding of activated 3MI metabolites to microsomal protein was inversely related to adduct production. There were both qualitative and quantitative differences in the formation of GSH adducts by lung and liver microsomes. In the presence of 2 mM GSH, the adduct was produced at a rate of 1.8 nmol/mg protein/min by lung microsomes but only at 0.1 nmol/mg protein/min by hepatic microsomes. The addition of cytosolic fractions containing glutathione S-transferase activity increased GSH adduct formation by approximately 30%. These results support the view that electrophilic 3MI intermediates are trapped by conjugation with GSH, and that organ-selective toxicity is primarily due to much faster rates of cytochrome P-450 oxidation of 3MI in the lung than in the liver.

The carcinogenic potential of anticancer drugs is discussed in the light of selected basic principles of chemical carcinogenesis. Anticancer drugs which act by alkylation and/or by binding tightly to DNA frequently cause cancer in experimental animals and may be carcinogenic in man. In addition, certain anticancer drugs act as cocarcinogens in experimental systems and augment the tumorigenicity of chemical carcinogens. Host determinants are important in chemical carcinogenesis. Many chemical carcinogens and anticancer drugs require metabolic activation by microsomal enzymes. Studies in twins have shown interindividual variation of drug metabolism in man is greater than intraindividual variation caused by exogenous factors. Therefore, certain individuals may be unusually susceptible to the carcinogenicity of anticancer drugs on a pharmacogenetic basis. Age is also a host determinant. At a given total dose level, age at first exposure to chemical carcinogens has been shown to be an important risk factor in experimental studies and in some epidemiologic investigations in man. Therefore, children may be especially susceptible to the carcinogenicity of anticancer drugs. These treated children have the potential of a normal lifespan; the latency period between initial exposure to a carcinogen and clinical evidence of cancer in man is long, usually 2-5 decades. The problems involved in extrapolating data of carcinogenicity in experimental animals to man are discussed. A single drug may have multiple consequences in experimental studies; for example, actinomycin D can act as an anticancer drug, an anticarcinogen, and a carcinogen. These uncertainities and the clinical results concerning second neoplasms following cancer therapy in both children and adults clearly indicate the need to follow carefully long-term survivors who have received cancer therapy.

An impressive array of evidence has been obtained during the past decade establishing correlations between specific DNA adducts and carcinogenesis. Many of the studies utilized organ specific differences in carcinogenesis to establish the correlations. More recently, we have investigated similar relationships between target and nontarget cell populations within the liver. Chronic exposure to methylating hepatocarcinogens predominantly induces hemangiosarcomas, whereas exposure to ethylating agents causes hepatocellular carcinomas. This cell specificity in carcinogenesis correlates well with the presence of promutagenic DNA adducts. In the case of methylating agents, the nonparenchymal cells accumulate O6-methylguanine whereas the hepatocytes do not. Exposure to ethylating agents leads to accumulation of O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine in hepatocytes. These differences reflect the ability of the two cell populations to repair O6-alkylguanine and the extent of purine and pyrimidine alkylation with methylating and ethylating agents. Hepatocytes of rats exposed to diethylnitrosamine for 28 days have four to five times more promutagenic DNA adducts (O6-alkyldeoxyguanosine and O4-alkyldeoxythymidine) than hepatocytes of rats exposed to nearly equimolar doses of dimethylhydrazine. Both O6-methylguanine and O4-methyldeoxythymidine are rapidly repaired by rat hepatocytes, while only O6-ethyldeoxyguanosine is rapidly repaired. Studies comparing the relationship between the induction of gamma-glutamyl transpeptidase-positive foci, hepatocellular carcinoma and promutagenic lesions such as O4-ethyldeoxythymidine will be useful in understanding associations between the molecular dosimetry of DNA adducts, initiation, and progression of hepatocarcinogenesis.

A class of soft alkylating compounds as potential anticancer agents was developed. The first examples include alpha-halo esters of various carboxylic acids. A new method for quantitative evaluation of the alkylating reactivity was developed, using a competitive alkylation reactivity was developed, using a competitive alkylation reaction, followed by NMR analysis of the reaction mixture. The method is sensitive and reproducible. One of the two selected soft alkylating agents, chloromethyl hexanoate, was found to have anticancer activity.

This article reviews the structure-activity characteristics, mode of action, pharmacokinetics and clinical utility of a group of chemically dissimilar antitumour agents which have as a common structural feature the N-methyl moiety. The importance of this feature is shown by the fact that molecules without a substituent on the nitrogen or compounds with N-alkyl groups other than methyl are usually inactive in experimental systems. This observation is supported by structure-activity studies with N-alkyl derivatives of s-triazines, triazenes, formamides, hydrazines and nitrosoureas. Representatives of these structural types which have found clinical application are, respectively, hexamethylmelamine, dacarbazine, N-methylformamide, procarbazine and streptozotocin. Mode of action studies have shown that dacarbazine, procarbazine and streptozotocin can give rise to species capable of methylating nucleic acid. This may be the lesion which produces antitumour activity. The mechanism of action of N-methylmelamines and N-methylformamide remains unclear. There is good evidence that, with the exception of N-methylnitrosoureas, host metabolism is prerequisite for activity with these agents. Although not pronounced, the clinical activity of N-methyl antitumour agents is useful, particularly as activity is not associated with severe haematological toxicity. Furthermore, responses may be observed in patients resistant to bifunctional alkylating agents. It is concluded that the drugs reviewed herein show a degree of coincidence in terms of their biological properties which may warrant a common classification. The term N-methyl antitumour agent is proposed.

The alkylating activity of extracts from several fish products with or without sodium nitrite in simulated gastric juice has been investigated. Some of the extracts had strong alkylating potency which may be due to the action of the formed N-nitrosamides. These compounds were not derived from nitrosation of methylguanidine and agmatine or from pyrolysis products of the processed fish. The results suggest an urgent need for a comprehensive investigation of nitrosation of various foods in simulated gastric juice.

The reaction characteristics of chlorothalonil with glyceraldehyde-3-phosphate dehydrogenase (GPDH), from yeast, (EC 1.2.1.12) were studied in vitro. Enzyme inhibition was related to the amount of [14C]chlorothalonil bound to the protein. Kinetics of enzyme inhibition was non-competitive for the substrate glyceraldehyde-3-phosphate (GAP) (Ki = 0.42 muM). Reversal of enzyme inhibition could not be demonstrated with the low molecular thiol dithiothreitol (DDT), although the thiol did protect the protein against the toxic action of the fungicide. Because 5,5' dithiobis-(2-nitrobenzoic) acid (DTNB) reduced the binding of 14C-labeled fungicide by approximately 90% it is postulated that chlorothalonil affects catalytic activity by reacting with the 4 sulfhydryl sites (cysteine-149) responsible for the binding of GAP. Certain reaction characteristics of the trichloromethyl fulfenyl fungicides with GPDH were found to be similar to those of chlorothalonil. However, chlorothalonil differed from those fungicides in that it did not react with non-thiol groups of either GPDH or alpha-chymotrypsin (alphaCT) and had a slower reaction rate with the GPDH. It is suggested that the differences in reaction rates of the fungicides are due to the molecular size and the chemical nature of the reactive toxiphores.

Methylation of horse heart cytochrome c has been examined in vitro with [methyl-14C]methanesulfonate (MMS) and [1-methyl-14C]-1-nitrosourea (MNU) as alkylating agents. Analysis of protein hydrolyzates by an automatic amino acid analyzer indicates that, at pH 9.0 with MMS, epsilon-N-monomethyl-lysine is found to be the only major methylated basic amino acid. On the other hand, the identity of the predominant basic amino acid residue which is [methyl-14C]-labeled by MNU cannot be determined at present. Peptide mapping of chymotryptic digests of cytochrome c after reaction with MMS reveals a lack of specificity in methylation of a specific lysine residue in this hemoprotein.

The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals. Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active. The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.

Muscarinic receptors have been characterized in smooth muscle and brain by the binding of reversible (e.g. atropine, quinuclidinylbenzylate) or irreversible (benzilylcholine or propylbenzilylcholine mustards) ligands. There is a close correlation between affinity constants derived from binding experiments and the affinities of muscarinic ligands for these sites obtained in pharmacological experiments on smooth muscle. Whereas atropine shows a single high affinity binding component (in subcellular preparations) several other ligands (QNB, ACh, oxotremorine) show multiple affinity binding. This indicated the existence of several types of binding sides which show selectivity toward certain cholinergic effectors. Most detergents inhibit the binding of ligands to the receptor site and therefore cannot be used to solubilize the receptor protein from the membrane. Treatment of brain subcellular membrane preparations with high salt concentrations (2M NaI) solubilize proteins which possess the muscarinic ligand binding properties observed in the membrane preparation. The affinities for muscarinic antagonists however are decreased, which suggests that a conformational change occurs in the protein upon solubilization.

The nature of at least three stages in carcinogenesis by chemical agents justifies the existence of a threshold dose: (1) a cytoplasmic stage during which most carcinogenic molecules are eliminated, (2) a nuclear stage during which certain DNA lesions are repaired and therefore cannot help to bring about mutations, and (3) an extracellular stage during which mutations are controlled for a long time by positive or negative epigenetic factors. All these findings are very difficult to collate. Since threshold doses cannot be detected by direct experimentation, the knowledge that a threshold exists is of little practical use and does not much alter the data permitting extrapolation of the results obtained with high doses of carcinogens. At the theoretical level only, this conclusion renders obsolete the opinion that all carcinogenic agents should be completely eliminated from our environment, and also the view that DNA needs only one hit from such an agent for a carcinogenic effect to be produced. The above conclusion also provides a more rational basis for the concept of a 'virtually safe dose' (VSD) or 'allowable daily intake' (ADI).

In vivo alkylation of Yoshida sarcoma cell DNA by 3H-labelled 1,2:5,6-dianhydrogalactitol (DAG) yielded N-7 monogalactitylguanines and 1,6-di-(guanin-7-yl)-galactitol, similar to the alkylated products obtained by in vitro reaction of DNA with dianhydrogalactitol in neutral solution. The ratio between monoalkylguanines and diguaninyl product was 2-2.5, slightly increasing with doses. Persistence of alkylated products in DNA was followed in function of time. There was no significant loss of either monoalkylated bases or diguaninyl derivative during the observation period i.e. 7-24 h after treatment. In contrast, the physical measurements of the amount of renaturable DNA showed a rapid opening of cross-links in the same period. Taking the presence of diguaninyl moiety as an indicator of cross-links in DNA, these two latter findings show an apparent contradiction which could be reconciled however by the mechanism proposed by Reid and Walker (Biochim. Biophys. Acta, 179 (1969) 179) for the removal of cross-linkage induced by HN2. Accordingly, one arm of the cross-links is removed, probably enzymically, leaving the DNA non-renaturable, while the other arm of cross-link is still covalently attached to the DNA molecule rendering possible the detection of diguaninyl moiety in DNA at some later time. This concept for the removal of cross-links from DNA seems to be supported by our results too.

The stoichiometry and kinetics of the anaerobic reactions between some thiols and derivatives of 2- and 6-methyl-1,4-naphthoquinones in water were measured using stopped flow spectrophotometry. The stoichiometry of the reaction with representative compounds was 1:2 thiol:quinone, a finding consistent with the formation of a hydroquinone as well as a thioether in the reaction. The first-order dependence of rate on thiol concentration, and the pH-dependent rate constants indicated that the thiolate anion was involved in the rate-limiting step, with rate constants at pH 7.6 generally increasing in the order glutathione (GSH) less than cysteamine less than dithiothreitol (DTT) less than cysteine. Despite the lower reactivity of GSH, the half-lives of the uncatalyzed conjugation reaction of these quinones at typical biological concentrations of GSH (e.g. 2 mM) ranged from about 2.0 to 20 s at pH 7.6 and 25 degrees C. The implications of these reactions in the use of naphthoquinones as potential bioreductive alkylating agents and as hypoxic cell radiosensitizers are discussed.

Transplacental carcinogenesis by N-ethyl-N-nitrosourea (ENU) was studied in patas (Erythrocebus patas) and rhesus (Macaca mulatta) monkeys. Repeated intravenous injections throughout pregnancy caused gestational choriocarcinoma in female patas monkeys and a variety of non-trophoblastic neoplasms in their offspring. Latent periods for transplacentally induced tumours varied from less than one month to more than ten years. One case of congenital neoplasm was observed. Certain kinds of neoplasms were observed only in transplacentally exposed offspring, and not in monkeys given the same carcinogen during juvenile or adult life. These included a variety of embryonal tumours, especially nephroblastoma, and tumours of the brain, mostly gliomas. Schwannomas of the peripheral nervous system were not observed in patas monkeys given ENU. One embryonal tumour yielded DNA that transformed NIH 3T3 cells in a transfection assay. Similar protocols performed in rhesus monkeys also yielded a variety of tumours in the offspring, including brain tumours; but in this species the most common embryonal tumour was pulmonary blastoma, and no choriocarcinoma was seen in adult females given ENU during pregnancy. Administration of N-nitrosodiethylamine to patas monkeys during pregnancy at first appeared to have had no effect on the offspring, but administration of phenobarbital beginning at four years of age resulted in the rapid appearance of multiple hepatocellular tumours. With regard to potential human risk from prenatal exposure to carcinogens, three conclusions deserve special emphasis: (1) the extreme susceptibility of the fetal primate central nervous system to certain chemical carcinogens, which confirms and reinforces what was previously known from studies in rodents; (2) the prolonged latency of transplacentally initiated epithelial tumours and the importance of subsequent postnatal exposure to promoting agents; and (3) the concurrent risk of transplacental chemical carcinogenesis in offspring and gestational choriocarcinoma in the mother, suggesting that gestational choriocarcinoma with its short latent period may serve as an epidemiologically exploitable marker for human populations in which transplacental carcinogenesis is likely.

This is a review of the work on molecular dosimetry carried out by several laboratories within the third Environmental Programme of the Commission of the European Communities. The molecular dosimetry project is a coordinated research project in which several laboratories cooperated on various aspects of quantitative comparative mutagenesis. This involved (i) development of assay systems for measurement of DNA adducts using radioactively labelled mutagens; (ii) development of immunological assays for detection and quantification of DNA adducts; (iii) isolation and characterization of monoclonal antibodies against DNA adducts; (iv) quantitative comparison of genetic effects of the model compounds within one assay system; and (v) extrapolation of genetic effects of the model compounds from in vitro systems to whole animal studies.

Diacetyldianhydrogalactitol (DADAG), a new alkylating hexitol derivative, was given in 30-min infusions for 5 consecutive days or as a single high-dose administration. The parent drug was eliminated in a biphasic manner, with a terminal half-life of 30-40 h. Dianhydrogalactitol (DAG), the main, pharmacologically active metabolite, appeared after a lag time of about 0.2-0.6 h. Its peak concentration was reached 1-2 h after termination of the infusion. The terminal elimination of DAG followed that of the parent compound. During the 5-day schedule slight accumulation was observed, and the plasma concentrations of both compounds approached the steady state. Over a dose range of 75-1050 mg/m2 the daily mean plasma concentrations of DADAG increased by only about 3-4 times. Dose-dependent expansions of the distribution volumes of the drug (Vc, V lambda, Vss) were observed. The behavior of DADAG and DAG in the body could be adequately described by a three-compartment open model. After equilibration the plasma levels of the parent compound and its metabolite were determined by the rate of return of DADAG from the peripheral compartment and its conversion to DAG.

An alteration in membrane transport is one of the most common mechanisms by which tumour cells become resistant to anticancer agents and represents one of the major obstacles in present cancer chemotherapy. A recent emphasis on understanding the mechanisms by which drugs are transported into cells should continue to assist attempts to overcome these problems. Resistance in certain instances can be overcome by modifications in the structure of a drug which increases lipophilicity. These successes have usually been obtained where well defined transport processes are present and specific deletions or modifications in the putative transport proteins are apparent, as in the case of classical antifolates. Simplicity, however, never seems to prevail and recent evidence indicates that cells can prevent transport of lipophilic drugs perhaps by modification of the chemical structure or properties of the plasma membrane, as in the case of trimetrexate. Some alkylating agents are transported by well defined carrier systems; for example, nitrogen mustard enters cells by the choline carrier, and melphalan uses two distinct amino acid carrier mechanisms. For both of these agents, tumour cell resistance is sometimes caused by transport defects. Resistance to antitumour nucleosides may be associated with impaired transport, and this has been demonstrated most clearly for cytarabine and fluorodeoxyuridine. In the case of antitumour antibiotics, although resistance is most often associated with a pleiotropic resistance phenotype in which the rate of drug efflux from the cell is increased, certain cases will be discussed in which resistance may be clearly attributed to defective drug uptake.

Radioactive alkylating 5'-[32P]-[4-(N-2-chlorethyl)N-methylaminobenzyl]-5'-phospham ide decadeoxyribothymidilate derivatives containing either free hydroxyl group (reagent I), hydrophobic cholesterol residue (reagent II) or polyaromatic phenazinium residue (reagent III) at 3'-termini were synthesized. The products were purified by HPLC and used for oligonucleotide-directed alkylating of DNA in isolated rat liver nuclei, Krebs-2 ascite carcinoma cells and L-929 murine fibroblasts. The uptake of reagent II by the cells was two orders of magnitude higher than that of reagent I and III. Intracellular alkylation of DNA by reagent II both in isolated nuclei and in living cells was about one order of magnitude higher than in the case of reagent I. The presence of phenazinium at 3'-termini of the reagent III leads to a sufficient increase of the alkylation extent compared to reagent I despite a quite low extent of its uptake by the cells.