Antimutagenesis and anticarcinogenesis: progress without molecular mechanisms

monitor

TIG - - Marck 1986

Overall, this was an interesting meeting* characterized by a manageable size, singlesessions only, interesting posters and ample time for discussion, both duringthe sessions and the social hours after the eveningsessions. However, although there was fairly balanced tine devoted to rep~'.,'tson experimentalwork in animals and c"lmiml intervention studies compared with reports on the molecular basis for genetic or carcinogenic effects, there seemed to be no meshing of the two approaches. No paper described an anticarcinogenic or ant~,utagenic effect and then gave the molecular basis for this. finding, Nevertheless, there were many interesting presentations, some of which are desm'bed below. The sessions began with presentations on the role of free mdi~ in chemical cardnogenesis 0~yor, Louisiana State University), purificationof liver cytochrome P-446 (Strobd, Houston, Terns), and the inactivation or activation of mumgens by catalase, poroxidaseand superoxide dismutase (Nagao, National Cancer Center Research Institute, Tokyo). A radical from cigarette smoke, identified by electro~ spin resonance as a semi~mdnone, bound to all types of DNA bases, and could be extracted into water if a sp'm-trappingagent were added. The half-livesof reactive oxygen as radicals varied from I0"° s in HO'to 10 s in RO0" or to days for tl~ q,uino~es found in cigarette tar. As for cytochromo P-

450, approximately 20 forms (moL wt about 76000; 678 amino adds) have been isolated and the amino acid sequences have been determined for some of them. There is considerable diversity in the levels and functions of the various forms. Peroxidases, especially those from horseradish, suppressed the mutagenidty of compounds from broiled foods; the catalase from rat liver cFosol decreased the mutagenidty of coffee (of which about 109~ is due to methylglyoxal and 5% is due to H,Oz); a preparation containing superoxide dismutase (SOD) enhanced the mutageniceffect of quercetin, of norharman and of

*International Conference on MechanismsofAntimstagenesisand Anticarcinogenesis, 6-10 October 1985, at the Universityof Kansas, Lawrence, KA, USA.

Antimutagenesisand anticarcinogenesis: progress without molecular mechanisms Elizabeth K. Weisburger Division of CancerEgoloRy, National Cancerlmsfihde, Beli~s~, MD 2O892, USA.

aniline, but a molecular explanation was not furnished. Bartsch 0ARC, Lyon) reviewed the types of compounds which in~it endogenous nitrosamine synthesis, the measurement of noncarcinogenicnitrosoproline as an indicator of nitrosamine synthesis, and application of this test to study smokers, betel quidchewers and specific popu~tions with a ~ n risk of stomach cancer. Although ascorbic acid inhibited formation of nitroso compounds in 40% of chewers of betel quid, in 60% it Mohn (Leiden, Netherlands) and Ketterer (Courtaukl Institute, London), described how, in some cases, ghtathiune may cause formation of more genoto~ic substances rather than de)mdcation products Gintathi~ne does not react well with hard electrophiles such as benzo(a)pyrene diol epoxide (BPDE) unless an enzyme also participates, but it reacts well with a soft electrophile such as N-acetyl-p-be~ imine. In the rat there are numerous glututhione lrandemses with a range of activities, and they can be induced by the usual enzyme inducers. Wattenberg (University of Minnesota, Minneapolis) reviewed the field of anticarcinogenesis with emphasis on: (1) blocking agents (flavones, indoles, barbiturates) which act largely by inducing various detoxifying enzymes so that active carcinogen never reaches a critical site; or (2) suppressing agents (retinoids, protease inbibitors, isothiocymmtes) which iubib'#,the cehSdarconsequences of response to carcinogens. A session on 'Avoidance of Spontaneously Occardag Errors in DNA' included several presentations on molecular genetics.

~) I ~ . Elsevier Science P ~ . ~ m KV., ArJstenlam 0168 - 952,qI~i~0.200

Echols (Berkeley) reported that the DNA polymerase m holoenzyme of E. coli has various subunits associated with different genes; however, the subunits do not have much interaction. UVtreatment may lead to lower fidelityin DNA repair, and mutagenesis may involve iubibition of recognition of lesions and editing of errors. E. coil go to a great deal of trouble to delete errors in genome duplication. Su (Duke University, Durham) deselegant tedmical approaches to study mismatch r~ ~. E:- ~;i..-Of @ of the genetic requirements for in mismatch correction, the wild type had the greatest relative activity, followedby mut H, ssb, mut L, mut S, and UVRD. He concluded there must be a communication mechanism between the misnmtcbed side and the non-mismatched side, but gave no data to support this conclusion. The excellent technical presentation 'A~letion and Oxidative Damages to DNA: Constitut/ve and Induc/ble Repair Systems' by Demple(Harvard)was part of a session on'Avoidance of Errors after DNA Damage'. Response of E. co/i to methyl~guan~ne (MNNG) v a ~ depending on the dose. Free r,dicals suchas O~-alsolead to damage, whichcan bo equated totbeamountdthymme~coland thymidine glycol in the DNA. Although H~Oz also damages DNA, it was not toxic if catalase syntbe~s was induced. E. coli hcking the XO3 gene were very sensitive to the effects of HzOz or bleomycin.Sekigachi(Kynshu University, Japan) compared inducible and regulatory genes in E. co//, suchasthe a/kAand u.,n= genes which are induced by MNNG or the ethyl analog, ENNG. MNNGwas also a good

inducer for the ada-/acz fused gene. The ada gene protein of apprommtely 39000 tool, wt had 354 amino acids and was pronated by methylnitrosourea (MNU). The/ac and ab~Agenes were repressed by MNU, but after treatment with methyl methanesulfomte (MMS) there was an induction or overproduction of p-aria and p-a/kA genes. Genes involved in excisionrepairinthe yeast Saccharomyces cerevisiae are UVRD and poi A; genes required are UVRD, UVRB, UVRC. Friedberg (Stap_ford) isolated five plasmids which confer host resistance. The Rad 3 UVRB genes have many amino acid sequences in common; Rad 3 protein may be a DNA binding protein. AlthoughRad2 gene was induced by DNA damage, Rad l and 3 were not. In £. coil, umuC and umuD mutants were produced by MNU treatment. The effects of the pKM101 plasmid depend on the rec A gene which plays at least two roles (Walker, MIT). Glickman (York University, Toronto) found that only 4% of spontaneous mutations were caused by ~sertion ofanion and there were not many G.T~A.T U'ansversions in the mutants examined; there was no correlation between the site of transitions and the number of events. Interestingly, slightly over half of the mutants had improved characteristics. The genetics and molecular biologyof mammaliancegs were covered in two sessions. Lambert (Cold Spring Harbor) found that in rat fibroblasts exposed to low doses of BPDE, there was increased replication of polyoma virus. Treatment of cells with BPDE also led to the appearance of a new protein which disappeared in about 24 h. H,Oz, sodium azide and bromodeoxyuridine were very effective in induction of cop/a, a heat shock protein, and a Drosophila retroviral element, after introduction into the cells. There may be a connection between ~'esponse to heat shock and the type of cop/e response in mammnlinq cells. Peilicer (New York University Medical Center) d e ~ the induction of T-cell lymphomas in ' mice with MNU or radiation, the activation of ras-genes by this technique, and the sequencing of the DNA fragments. Inoue (National Institute of Ge~tics, Mishima,Japan) used cytological and biochemical studies on the 59

erspectives mouse mutant 'wasted' in an effort to findan animalmodelfor Atag/a tela~tas/a. Although the mutant has some traits characteristic ofAtm'/a, inothers it differed. Further studies onthe response of the mutant skin fibroblaststo UVandcarcinogens are needed to establish it as a sound model. Thompson (Livermore National Laboratory, Cali. fornia) developed mutants of CHO cells which were defective in the incision step of nucleotide excisionrepair and inthe excision of bulky adducts. Hybrid cells were constructed by fusion with human lymphocytes in order to isolate the human repair sequences. Kuroda (National Institute of Genetics, Mishima, Japan) found that with Chinese hamster V79 cells, compounds such as aniline, o-, m-, and

TIC, - - March 1986

p-chloroaniline, nitrobenzene, hexachlorobenzene and CHCls, which were all negative in the Ames Salmonella test, induced 8.a~@uanine - but not ouabainresistant mutations. Vitamin C inhibited the 6-thioguanine resistant mutations induced by EMS in these cells.

Three sessions were devotecl_ to 'Mechanisms of Carcinegenicity and Anticarcinogenicity'. Stichand associates (Vancouver, Canada) have underway efforts to reduce the genotoxic damage in the oral mucosa of betel quid/ tobacco chewers. Criteria for selection of specific population groups, the non-invasivemicronucleus test used to examine shed epithelial cells, and preliminary preventive trials with vitamin A and carotene were described. The protective action

of calcium salts against the toxicityof bileacidsand ricinoleic acid was discussed by Brace (Ludwig Institute, Toronto) and Wargovich(Universityof Texas, Houston): another reason to drink milk! Borek (Columbia University) reported that lack of thyroid hormone makes cultured !~_.ster ~l]s re~ctory to radia. tion, benzo(a)pyreneor Ki-MSV virus. Response was restored by addition of thyroxine to the culture medium. Transformation and promotion in these cells were inhibited if poly(A'rP). ribose were suppressed. AIthough cells from Xovdemm p/smentosum (XP) patients do not repair well (or only slowly) the large DNA adducts from carcinogens such as aflatoxinor benzo(a)pyrene, they repair norreally after exposure to MMS Or

MNNG which lead to small adducts. The relationship between eight complementation groups and the range of UVinduced unscheduled DNA synthesis in XP.hybrida was determined; ranges varied from 0 to 100~ but were generally in the range 10-.40~ (Cleaver, San Francisco). Benedict (Children's Hospital, Los Angeles)indicated that retinoblastomais associated with a probable 1~14 deletion and that both gas and myc oncogenes are necessary for trm~onmtion; in cells from patients, there was an increase of N-mycgene. Many other interesting and relevant papers were presented, mostlyou the beneficialeffectsof vegetables or plant compounds, but they were somewhat removedfrom the fieldof genetics.

Molecular mechanisms for gene conversion in higher cells

The accumulation of structural data on eukaryotic genes has provided new insights into genetic variation in families of repeated sequences and mnltigene families. Much attention has been given to gene families such Genc conversionin highereuka~yotesis a conceptsofar devoidof molecularsubstance. It as giobin, actin, myosin, etc. in a iS ShOWnhere that two classes of hypothetical mechanisms, based on singleo or double° variety of organisms, and a large strand instructed repair, lead to different predictions. The inigalion of conversion number of mutations causing, or evonls in relagon with recombina6on or mismatch correction is discussed. associated with, human hereditary diseases, particularlyin the globin genes, have been remains unaffected, while gene A, which shares analysed at the nudeotide level In addition, gene families extensive homology with B, receives a block of B encoding polymorphic products, such as immunoglobulins, sequence and undergoes variation. There are now a T cell receptors, class I and class H I-I-2and HLA antigens number of examples in which nucleotide sequence data in mouse and man, have been studied intensively. are compatible with the interpretation that variation Together with analyses of repeated sequences, and occurred in precisely this way. Examples include rapid progress in simpler organisms such as yeast and exchanges between the human [3-giobin genesZ, the Drosophila, whose genetics are more easily studied than Cy2a and Cyb genes of the mouse immunoglobuEns those of mammals, the available data have led to an heavy chains=, the K° and Q10 mouse class I H-2 genes, important move in the concepts of gene variation in creath]g_ the bml mutation4 and the human ~-giobin mul~ene families. In essence, the conviction has genes ~. In most cases, though, the sequences show the spread that the random occurrence of single point trace of an ancient event and the parents are not mutations is neither the only, nor perhaps the major ~vailable for analysis, so that it is difficult to rigorously route to gene variation: instead, it is now believed that establish the non-reciprocal character of the sequence members of a multigene family occasionally exchange transfer. However, direct proof for conversion has been information, and often do so by transferring blocks of obt~ed in mouse L cells transfected with two nucleotides in a reciprocal or non-reciprocal way. contiguous neo genes carrying specific defects: in certmn configurations, acquisition of resistance to the antibiotic Gene conversion 13418 (which requires a functioml neo gene) was due to Gene conversion formally describes a non-reciprocal gene conversion and occurred at a frequency of about 5 transfer of genetic information observed best pheno- × 10-° (Ref. 6). Such observations have been typically in tetrad and octad forming microorganisms, reproduced in other systems 7,s. such as yeast and Ascobolus. Gene conversion, as Although gene conversion takes place in higher cells applied to multigene families of higher organisms, has and organisms, as it does in lower e~mryotes, very little become popular since BaltimoreI called attention to the is known about the molecular mechanism(s) involved. As potential of a non-reciprocal transfer of a block of ]9 and others I° emphasized earlier, the term gene sequence to generate homogeneity and/or variation. conversion, used as it is in the higher etdmryotes without The term has spread as a metaphor which is largely too much caution, may become misleading; as long as it irrelevant to its initial genetic background. I shall use the remains a fomml concept with no mechanistic following definition: gene conversion describes any background, it may indeed lead to oversimplified views. process in which gene B acts as a sequence donor and It is, therefore, important to give molecular substance to 60 ~) 1986, Elsevier Science PublishetsB.V., Amsterdam 0168 - 9525/86/$0.200

Philippe Kourilsky