Short-term tests in screening programs of environmental chemical carcinogens

Exp. Path. 22, 187- 202 (1982) Friedrich Schiller University J ena, Institu t e of Path ology (Head: Pr of. Dr. se. med. Dr. h. c. F. BOLCK)

Review Short-term tests carcinogens

In

screenmg programs of environmental chemical

By E. AMLACHER (Received May 25, 1982) A dress for correspondence: Dr. rer. nat. hab il. E. AlILAC HER, Path ologisches In sti t ut der FriedrichSchiller- Universitat, Ziegehmihlenweg 1, DDR - 6900 Je na Key wor d s : mutation, somatic; carcin ogens, mutagenicity ; assays, short-term; carcinogenicity tests; Bacterial mutation t est (Bl\1T) ; Al\lES t est; Cell transformation test (CTT) ; DNA-cell-binding assay (DCll A); Unscheduled DNA-repair t est (USDT); Thymidine screening syst em (TSS) ; met abolic aeti va tio nin vitro; t est accuracy; carcinogenicity, phar maceutical products; carcinogenicity, agricult ura l products ; bioassay; economical expenses

Summary Fo llowing an intro ductory m ent ion of t he genet ic bases of can cer inducti on (t heory of soma t ic muta ti on) a survey of the diver se shor t te r m bioassays t o t est carcinoge nic chemi cal compo unds is given . Within the large nu mb er of hitherto pu bli sh ed t est s, fiv e of them are acce nt u ate d in particular: Th e Bac t erial mutation test of AMES (BMT), the Cell transform ation test of S TYLES (CTT) , t he Uns cheduled D NA repair t est (U SD T), the DNA-Cell-binding assay of KUBINSKI (DCBA) and t he author' s own nuclear D NA sy nt hesis (t hy midine in corp oration) inhibiting scree ning syst em (T88). Each of these five te sts is present ed individuall y a nd afte r t ha t t hey a re compared a nd crit ica lly reviewed , as is done in p ar t icul ar conce rn ing t he l b l E S assay.

I ntroduction Th e high level of the human cancer bu rden is r elated t o the exposition t o u, large nu mb er of manmade carcinoge ni c age nts . The poss ibi lity of genoto xic D NA dam agin g actions of these compo unds promp t ed st udies in t o me t ho ds of establishing the carcinogenic pot ential of such che micals by aid of shor t-t er m scre ening as sa ys. Every ye ar countless newly produced che mical compounds are introduced into the hu man environment as medi caments, pe sticides, food additives, cosmet ics and ot her industrial produ ct s. A n otable number of t hese is ab le to induce mu t at ive changes in t he genet ic pool of humans, animals a nd pla nts. Th e fun dament a l con cept of somat ic mu t a t ive cellular a lt eratio ns ca usi ng mali gn ant t ransformat ion was postulated by BOVER] (1914), TYZZER (1916), BAUER(1928) and again in close in vestigati ons by BAUER(1963 ). Th e t erm " somat ic mutat ion" was int ro duce d by TYZZER, whil e BOVERI pointed out t he rol e of chromoso ma l delet ions leading to ca nce rous cellu lar changes. Th e concep t that macromolecul ar da mages in th e nuc lear (a nd mitoc hon drial? ) DNA cause s t he (mut ative) in itial step of ca rcinog enesis r eceived ennvincing su ppo rt in the last years. Th e degree of alterations of the geneti c informatio n is dependent a lso on DNA repair reserves. A mut at ion can be the result of mispairing or in com plet e r epair of a part of t he gene t ic rel ev ant DNA segm ents. Of 14*

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great interest in this is the synthetic phase of DNA (Suss and MAURER 1968; BERMAN et al. 1978), because of their enhanced sensitiveness in the interference of carcinogenic agents with the reduplicative macromoleeules (errors in the identical reduplication). The majority of short-term tests to detect carcinogenic potentials is founded on the leading idea of somatic mutative induction (mutation) of the chemical cancerization and were based on empirical observations in animal and human carcinogenesis. The mutative changes can be classified in two general types: 1. Molecular alterations (lesions) 1.1. DNA base pair mutations by substitutive mispairing of bases 1.2. Frameshift mutations of the genetic code through deletion or addition of bases, resp. as a consequence of intercalation of carcinogenic molecules in the frr.me of bases sequence of DNA 2,

Aberrations of chromosomal segments (deletions, duplications, inversions, translocations) as a result of alterations of DNA, one of the primary targets of carcinogenic agents. DNA alteration can lead to chromosome-type aberration before beginning and to chromatid-type aberrations during or after DNA synthesis (S phase).

Although the most carcinogens are mutagens (lYlILLER and MILLER 1971) and their initial activity is directed towards the alteration of the DNA bases sequence or frame, there are nonetheless some chemicals which apparently do not act in this way (promoters, carcinogenic hormones, solid-state carcinogens l. Obviously it is of greatest interest to relate the carcinogenicity of a chemical to its structure and to prove the precision of a bioassay with regard to its ability to select carcinogens in a group of neighbouring chemical compounds. Carcinogenesis seems to be a multistep process. depending on individual genetic constitution and the constellation of the genetic and environmental substrate. Two steps in carcinogenesis are essential: the carcinogenic initiation and the malignant growth promotion of the affected cells. Initiation involves the alteration of DNA, and occassionally a single low dose of a chemical carcinogen or It mutagen n: II produce a change in the sense of a somatic mutation (cp. SORS\ 1980). The problems of promotion will not be discussed in this presentation. "Long-term animal carcinogenicity tests are unable to keep up with the number of ehemicals requiring testing" (STYLES 1980). Therefore the variety of recent short-term tests based on mutative or DNA damaging alterative events will be briefly discussed in the subsequent essay. A survey of short term assays to detect potential carcinogens is given in table 1.

Presentation of [ioe effeehve short-term assays In the following survey five of the in our opinion most effective short term tests are discussed. BaeterialMutation Test (BMT, AMES, MCCANN) The test was developed on the basis of investigations performed by AMES, DURSTON, YAMASAKI and LEE (1973). AMES, MCCANN and YAMASAKI (1975), MCCANN, CROl, YAMASAKI and AMES (1970), AMES, MCCANN and SA WYER (1976), MCCANN and AMES (1976), SUGIMURA, YARAGI, NAGAO, TAKEl'CHI. KAWACHI, HARA, YAMASAKI, MATSUSHIMA, HASHIMOTO and OKADA (1976), ANDERSON and STYLES (1978), AMES (1979), HOOPER, HARRIS and AMES (1979), McMAHON, CLINE and THOMPSON (1979), RINKUS and LEGATOR (1979), CHELl, DE FRANCESCO, PETRl'LtA, Mct'ov and ROSENKRANZ (1980) and many others. B. N. AMES (1979): "This work, and that of others, has strongly supported the theory that most carcinogens act by damaging DNA. The Salmonella test and other short-term tests that have been developed for testing chemicals for their ability to interact with DNA or for mutagenicity are being widely used and should help in solving some of the problems that cannot be adequately approached by human epidemiulogy or animal cancer test alone .. .", •

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Table 1. Survey of most of the published tests to determine carcinogenic pot ential. In heavy type: assays whi ch hav e a accura cy exceeding 90 per cent and will be closely repr esented in our essay 1.

Mutation-evidenced tests I n vitro

• Ba c te r ia l m u t a ti 0 n te st ( B )IT). I n vitro metab olisati on of t he te sted compound by aid of a n auxiliary hom ogenate of isolat ed mammalian liver rnicrosomes. Demonstration by forward and reverse mutati ons. 1.2. Yeast mutation (gene mutati on, gene conversion, mitotic recombin ati on or non-di sjun ction). 1.3. Gene mut ations in mali gnent cell cult ures (lymphoma cells). 1.4. • Mammalian cell tr ansformati on t est (CTT) using mamm alian cell cult ures and liver microsomes for the met aboli sati on of seconda ry carcinogens un der st udy.

1.1.

I II vivo(aid-t ests) 1.5. Dominant-leth al test in mice a nd ham st ers. 1.G. Heritable- translocation test in ma mmals. 1.7. Sex-chromosome loss and non-d isjunction in mammals. 1.8. Specific -locus method in mamm als. 1.9. In vivo somatic mutation t est (mouse hair spot test). 1.10. Drosophila melanogaster test. 1.11. Host mediated assay.

2.

Non mutation expressed but DN A alterative biochemical and auionuiicqraphical tests Biochemical tests

2.1.

Whole DNA synthesis (growt h) inh ibition in dividing cell popul at ions influenced by carcinogens. Appli cabl e in syn chron ized growt h-stimula ted cell cult ures a nd in ect omized organs (liv er, kidney, pan creas). 2.2. DNA dam age test by quan tificati on of single strand breaks in whole mammalian organs after long term 3H-t hy midine lab eling of DNA a nd subsequent applicat ion of carcinogens t o th e test ani mals. 2.3. • D N A- ee l l- bin di n g a ssu y ( D C HA). Bacterial cult ures or isolat ed mamm ali an cells in culture were brought in conta rt wit h carcinogens and with isolat ed purifi ed [32P]-phosphate label ed DNA. Thi s DNA read s stron gly with cellular macromol ecules (DNA, RNA , proteins) producing specific adducts . The attachment of lab eled DNA in creases in t he presence of carcinogens. 2.4. • Un s ehe d ul e d D NA r ep ai r test ( U S D T). Enhan cement of unscheduled DNA synt hesis in HeLa "ell cult ures aft er reacti on with carcinogens in pr esence of liver microsomes or in prim ar y hepat ocyt e cell cult ures , Subsequent scintillometric eva luation of t he level of isolat ed USD. (The a utora diographic eva luation by silver grain roun tin g is also possible).

2.5. 2.0.

3.

Autoradunraphical tests DNA repair test as mention ed a bove in :!.4. • Nu cl ea.r DNA s y n th e s i s i nhi bi ti o n te st. (Thy midine in corp oration inhibiting t est, th ymidin e screening system TSS). Auto radiogra phical demon stration of decrease in 3H-thymidine in corporation into nuclei of kidne y and liver of suckli ng mice after i.p. injection of carcinogens.

Miscellaneous histological, cytological and biochemical tests

3.1. Sebaceous gland test. 3.2. Micronucleus test. 3.3. Pro mot ing activity t est. 3.4. Convent ional met aphase a na lysis or sister chromatic excha nge 3.5. Chromosomal dam age (cp. th e poin ts 1.5-1.10). 3.5.1. Mammalian bone marrow inv esti gation. 3.G. Degra nulation t est of rough endoplas mat ic reti culum. (The publi cations consult ed for t he survey in th is t able ar e cite d in th e lit erature index of t his work ).

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" Ou r work started as an offsh oot from our ba sic research on th e molecular biology of Sa lmonella ba cteria. We were st udy ing how genes ar e switc hed on and off in bact eria in response t o th e presen ce of th e a mino acid histidine in th e gro wt h medium and the effect of mutati ons whi ch p erturbed this control mechani sm. We were using a large collect ion of histi d ine-requiring ba cterial mutan t s .. . In 1964 we began t o develop a t est syste m for dete cting mu t agens using these mu t ant s. Dur ing th e course of t his work, and ot her work on the theory of fr am es.hift mutagenesis, we beca me conv inced th at t he essential pr operty of most carcinogens was th eir mutageni city. Th e id eas . . . had made a maj or contri but ion t o the underst anding t ha t many carc inog ens must be conve rte d by enzy mes in liver or ot her ti ssues t o an ac tive (electro philic) form th at is the t rue carcinoge n (and mu t agen) . . . We thus added mamma lian liver ti ssu e on th e t est to pr ovide a first approximati on of mammalian met ab olism ". " The t est is don e by combining on a petri plate th e compoun d to be t est ed, about 1 billi on Salmonella bact eria of a par t icular te st er strain (seve ra l diff er ent histidine-r equiring mutan t s are used), and hom ogeniz ed liver fro m rodents (or hum an auto psy ); afte r in cu bation at 38°C for 2 days, the numb er of bact erial reve rta nt colonies is recor ded. E ach colony is compose d of th e descendants of a bact erium t hat has been mutated from a defective histidine gene t o a fun cti onal one. Normally. om' test s doses of a chemical in a series of plates - t he plate t est and quantitative dose-response curves are generated. Th e curves are almost always lin ear , wh ich suggest s that, at least in t his syste m, thresholds ar e not common. Mutagens can be det ect ed at low doses, in some eases in nanogram amounts". At pr esent (sit uat ion of March 1976) ab out 300 carcinogens and no n cnrc inogens of different chemical t ype s were tested by AMES, MOCANN and coworkers in th e Salmonella micro som e t est. Th ere is a correlation of 157/175 betw een carcinog enicit y and mutagen icit y that mean s 90 % of the carcinogens wer e also mu tagenic in th e BMT includin g a great par t of kn own human carcinoge ns. F ew "noncarci nogens" show any degrel' of mut agenic (~et iv i ty (MCCANN et a l. 1975). Carcinogens negat ive in t he t est and a ppare nt rd ~ l' positives are discussed with resp ect t o t heir dam aging (m utative) effec t on D lXA. In recent add it ional assays by Md fAll ol'\ et al. (1979) 855 test chemicals were in vesti gat ed in J 0 test er st ra ins using a new modificati on of th e A)IES BlVIT. The chemical com pou nd s were t este d in t his assay ove r a 10,000-fold conce ntra tio n gradient, both wit h and without met abolic activatio n. 182 of 855 chemi cals were found to be mutagenic in one or more of th e te ster st rains. 20 0 0 of 299 chemicals used in chemica l manufacturing or lab oratory synt h esis gave a posit ive respo nse in diff erent bact erial st rains. Wit hin 361 organic chem icals synt hesized as pote ntial ph arm aceuti cal or agricult ural product s only 8 % were fou nd t o be mu t agenic. III rletecting fra meshift mu tagens t he S almonella iyphirn urium strai ns TA 98 a nd TA 1538 proved to be very efficient. The IIIOSt sensit ive t est er st ra in was TA 100, det ect ing 142 of 182 mu tagens (de teetion of bot h base su bst it ut ion and fram eshift mu tagens). H owever th e strain '1';\ tOO was not suita ble for t he specific ident ificat ion of base substi tutio n mut agens. Th is mut agen s were mor e rel iabl y det ect ed by the Escherichia coli t est er st ra ins WP 2 and WI' '2 uvr A:'. RIl'\KUS and LEGATOR (19791 ana lyzed 465 known or suspected carcinogens with regard t o t heir correlat ion with mut ageuie act ivit y in the Salmonella iyphimurium system. Th e auth ors acce nt uat e thct the " inno vati ve use of microsomes t o stim ulat e metabolism has not cha nged the faet that in rilro activation cannot duplicate fai t hfully the metabolism that occur s in vivo. This shortcom ing will exp res s itself b y the pro duct ion of fdse negatives and p ossibility fa lse positi ves during mutagenicit y scr eening. This asser t ion is al so borne out by a r ean alysis of the ability of known a nima l carcinogens t o cau se mu t ati ons in th e genera lly recognized pr emier in uiir« system. t he Salmonella-S 9 system. Alt hough pr evious st udies have suggest ed t ha t a high percentage (85 '\ .) of all ca rcinogens will be mutagenic in this syste m, with no indi cati on that Ia lse negatives are associated wit h certain chemica l ty pes, t hese findings are of un cert ain pract ical va lue du e t o t he limi t ed nu mber of chemica l t yp es th at were considered" . (c p. Discussion j. Th e analysis by R IJXK Di and L F,(L\ T O R uf 465 compounds with defined or susp ect ed carcinogenicity ind icated th at the testing in th e pa st ha s concentrated on certa in chemica l types an d t hat some categ ories of carci nogens ex hibit spe cific correlat ions " t ha t are un satisfact orily

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low by any standard". To optimize the testing of bactericidal, volatile or DNA-cross-link chemicals non-standard procedures are necessary. Following RINKUS and LEGATOR the 61 false negatives from 465 substances tested in the AMES assay probably arise for two reasons: 1. The inability to devise an 1:n vitro activation system with reliably standardized use. 2. The inability to detect the entire scale of cancerizing mutations. The last mentioned affirmation seems to be exaggerated with regard to the general purpose of all effective short-term assays employed at present (cp. Discussion). Following AMES et al. (1975) the expenditure of testing one chemical compound in the BMT amounts to 200-300 US-dollars. An exact specification of the cost details is missing in the above mentioned paper. • Cell transformation test (CTT, STYLES, A"DERSON) Malignant cell transformation in vitro was reported in the past years by DI MAYORCA, GREENBLATT. TRAUTHEN, SOLLER and GIORDANO (1973), MISHRA and DI MAYO RCA (1974), EVANS and DI PAOLO (1975) and by KIRKLAND, HARRIS and ARMSTRONG (1975). BOUCK and DI MAYORCA (1976) emphasize the somatic mutation as the basis for malignant transformation of cultured cells by chemical carcinogens. STYLBS (1977, 1978) describes a method for detecting carcinogens using mammalian cells in culture (Cell transformation test). Following STYLES (1977. 1978) baby hamster kidney cells (BHK-21/CI 13) were used to test 120 compounds, human lung cells rWl-38) to test 107 compounds and human liver cells (Chang) to test 17 compounds (cp. PURCHASE et ell. 1978). Additionally also the polyoma virus transformed cell line BRK 21 Py Y was used. The cell cultures were exposed to tested compounds in liquid tissue culture medium without serum, containing rat liver postmitochondrial supernatant and cofaetors (8 9-mix) to aid metabolism. The assay is based on the survival of cells after exposure to the chemical under study. The survival was assessed by cloning in liquid growth medium. The transformation frequency was stated due to the ability of transformed mammalian cells in culture to form colonies in soft (semiliquid) agar. A dose response curve for survival served for determination of the LC50 of the compound. Additionally a doseresponse curve for transformation Wi'S constructed. A 5-fold increase in transformation frequency at the LC50 was regarded as positive test result. The method may be used for testing gaseous compounds mixed with air, using cell monolayers overlaid with serum-free medium and S 9-mix. The exposed cells were trypsinized, resuspended in growth medium and then investigated for survival and transformation (for details cp. STYLES 1977). To dose the replicate cell suspensions the tested compounds were dissolved in DMSO at various concentrations. The concentration of the solvent was 1 v [v, For transformation the cells were incubated at 37°C in 200 ml of growth medium in glass roller bottles 1 I in of cells were confluent. At this stage they were trypsinized and revolume until about 90 suspended, 106 cells/ml in Medium 199. 1 ml portions were distributed into sterile MCCARTNEY bottles and to each was added 10 ml of S 9-mix and 10 ;ul of test compound solution. The suspension was incubated for 4 hours in an orbital shaking incubator. After incubation and centrifugation each pellet of cells was resuspended in growth medium to which molten p,gar solution was added. The thoroughly mixed cell suspension was poured into petri dishes. When the agar was allowed to gel, the suspension was treated with the mixture appropiate to the cell type and incubated for 14-21 days (37°C in a humified CO2 incubator). The transformation was quantified by rid of an inverted microscope at a 20-fold magnification. STYLES (cp. PURCHASE et al. 1978): "The cell transformation assay was, with the selection of accurate in determining carcinogenic or compounds tested, and the use of 2 cell lines, 94 non-carcinogenic activity. Of 120 compounds tested, only 11 were detected by one cell type alone; furthermore, if one eel] type only is considered, the predictive figures are only slightly reduced (88 for WI-38 and Chang, 91 '/0 for BRR 21). These results indieate that the species or organ of origi n of the tester cells is of no significance in this assay, and that one cell type offers no great predictive advantage over another ... " (cp. Discussion). 191

An economical evaluation 01' th e test costs is not presented by STYLES (PURCHASE et al. 1978) and also not by ANUR ESON(1979). • U nsc he d u le d D N A r ep a ir te st (CSDT) The induction of unscheduled DNA (USD) in mammalian cell cultures specially in primar y rat hepat oeyt es has been suggest ed as a possible short-term sereening test for carcinogenic agents. STICH and SA]'; (1970) reported on chromat id anomalies accompanied by repair DNA synthesis in mammalian cells exposed to derivati ves of 4-nitroquinoline-1-oxide. These investigati ons were cont inued by STICH, SAX and KAWAZOE (1971) and by STICH, KIESER and LAISHES (1973). SAN and S1'I<'I1 (1975) proposed th e DNA repair synt hesis in cultured human cells as a rap id bioassay for screening chemical careinogens. In similar investigati ons using primary rat liver cell cultures SA;Il and WILLIA MS (1977), WILLI AMS (1974, 1976, 1977, 1978), YAGER and MILL ER (1978) and }h ClL\LO POL:LOS, SATTLER, O'CONNOR and PI1'OT(1978) demonstr at ed the usefulness of induction of lS D as an indicat or of carcinogenic act ivity . For quanti ficati on of th e degree of I'S]) induction liquid scintillation counting was employed by MARTI N, McDERMID and (;A RNER( In77, HI78), whereas WILLI AMS (1976, 1977, 1978) and recentl y BRAMBILLA, CAV.\ :11:\ A, CARLO, 1"1:\01.1.0, Sn ABA, PARODI and BOLOGNESI (1979) report ed of results obtain ed hy aid of aut oradiographically silver grain count ing. A crit ical review comparing thr ee in vitro assays for carcinogen-induced DNA damage and consecut ive DNA repair (strand break assay, liquid scinti llat ion assay, autoradiographic assay) was given by PRO BST, HILL and BEWS EY (1980). Comparing the hitherto published test results obtained with th e USD assay on 47 well known carcinogens and 10 nnn-carcinogens using primar y cultured hepat oeytes by WILLIAMS (1976, 1977, 1978). :MA RTIX , i\1<' DEHMIll and GAR NER(1978) and by PROBST, HILL and BEWSE Y(1980) we found : 4 of 47 tested carcinogens gave a false negat ive result, th e aeeuracy of th e USD-test amount s to HI " " , t hat means 4;3 of th e teste d carcinogens were correct ly identif ied. 'Within 10 t ested non-carcinogenie compounds one was found to react in a false positive sense, that means the aecufltcy corresponds t o 90 ° ", The number of t est ed non-earcinogens fails to be representa t ive and the testi ng of addit ional chemicals is needed t o prove th e reliabilit y of this assay. It is generally notieed t hat t he effectiveness of th e USD-test depends on th e concentrations of t he app lied chemicals an d of the employed cell line in culture. Additionally, in general, compounds requiring metabolic act ivat ion elicit a lower response t han direct acting agents do. In some eases metab olizing enzymes are needed to enhance th e sensit ivity of tre at ed cells. l\lAHTI~ , l\IcDER}I1 D and (;ARNER( I\)78): " The major disad vantage of mammalian (eell cult ure!) short-term screening assays compared wit h bacterial mut agenicity assays are th e longer t ime required to t est each «ompound and t he reduced sensit ivity. Auto radiogra phy of treated eells has been the meth od of detection most widely used, but it is a rath er length y and somewhat t edious procedure. The use of scintillation countin g has reduced the time involved and is a less demanding tech nique". An economical evaluation of th e costs and the durati on of th e assay procedure is also lacking in the puhlications concerning th e USD repair test . • DNA- Cell-Binding As sa y (De BA, KUBINSKI) This test is based on invest igati ons effect uat ed by KUBINSKI and eo-workers (KUBINSKI, OPARA-Kl'BINS KI and SZY BAl.SKI 19GG, KL' BINS KI and SZYBAI.SKI 1975, KUBINSKI, MORIN and ZELOT IN 1976, Kr RDls KI r nd !\lORI:'> 1978, KUBI NSKI 1979, KUBINS KI, GUTZKE and OPA RA-Kt:BINS KI 1981). The following quotations are taken from the publicati on of H. KUBINS KI, G. E. GUTZKE and Z. O. KUBI NSKI (1Iut. Res. 89. 95---136, 1981): " The DCB assay is based on th e observa tion t hat nucleic acids, including J) NA, form complexes with prot eins and with ot her nucleic aci d molecules in th e pre, enee of act ive carcinogens. The format ion of such artificial nucleoprot eins and other nucleic ar-i d adducts may be recognized by a variety of analytical techniques: complexes produced in living cells are detect able by th e decreased yields of DNA recovered int o wate r phase during cold phenol ext ract ion of t he cellular lysate and by th e alka-

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line elution test ... an inactive carcinogen may be activated by appropiate enzymes and, again, such artificially induced complexes may be detected in most of these experimental systems". "When certain eukaryotic cells, such as Ehrlich ascites tumor, human HeLa cells, isolated and suspended mouse liver cells (unpublished), or prokaryotic cells such as Escherichia coli are mixed with nucleic acids in the presence of ultimate carcinogens (or in the case of carcinogens which require enzymatic activation, together with an appropiate activating system), the nucleic acids become strongly attached to the cells, This reaction is time-and concentrationdependent. That proteins arc the most likely target molecules in the cellular membrane has been shown by experiments in which DNA binding was abolished following a short exposure of cells ... to proteolytic enzymes ... ". The DCB assay is therefore an inoiiro system in which extracted purified bacterial DNA was labeled with [32P]-phosphate and mixed with a bacterial or animal cell culture suspension in presence of the chemical compound under study and in some eases by addition of various ingredients (lysozyme, activating enzymes) and/or of the AMES S 9-mix (liver postmitochondrial supernatant and culactors). In addition a "positive control" (with cells, DNA and the alkylating methyl methansulfonate) and a "negative control" (with only the cells and the DNA) were performed. For activation of secondary carcinogens to their ultimate form the mammalian liver extracts (8 9 fraction) were prepared as described by AMES et al. (1973) and used after repeated freezing and thawing to destroy the troubling enhancement of cellular DNA binding caused by these liver microsomes. DNA and RNA exposed to active carcinogens and added to the cell suspension, strongly react with cellular macromolecules, producing nucleic acid-nucleic acid and nucleic acidprotein adducts, The latter group of adducts included complexes with protein present in both prokaryotic and eukaryotic cell membranes. Increased attachment of labeled DNA to the intact bacterial and cultured animal tells was seen in the presence of active carcinogens or carcinogens activated by extracts from mouse and rat livers. The DCBA is relatively insensitive to minor errors of the experimenter but sensitive, however, to major changes in pH. Within 280 investigated chemical compounds and mixtures 65 known carcinogens and 70 known non-carcinogens were tested. The DCB assay was positive in 62 cases of carcinogens (95.4 agreement with the animal data) and negative in 67 cases of tested non-carcinngens (agreement in 95.7 '; 0 of cases). Economical considerations: Equipment for an exacting biochemical laboratory is needed including the subsequent amortization of this capital. The cost of testing one substance amounts to 100 dollars (U.S.) in 1980prices. The DCB assay is eompleted within 2-3 hours (including time needed to prepare the sample). The number of involved investigators and their salaries were 110t stated. Limitations: The test cannot be performed away from a well-equipped laboratory. The testing of gaseous carcinogens is not practicable. Some "false negatives" are probably due to deficiencies in the aetivating system. The test can be considered as a predictor of potential carcinogenicity . • Thymidine incorporation inhibiting screening system (TSS, AIIILACHER) The so-called thymidine screening system is based on the exclusive autoradiographically demonstrable phenomenon of nuclear DNA synthesis suppression by chemical carcinogens, revealed as a decrease of silver grain number over nuclei in the S phase (TOMINAGA, LIBBY and DAo 1970; ORD 1976; AMLACHER, DANz, 8TftLER and RUDOLPH 1977; AMLACHER and BOLeK 1977; AMLACHER und ReooLPII 1978; AMLACHER and ZIEBARTH 1979; KRAMPITZ and SEEMAYER 1979; SEE;\L\YER and KRAlIIPITZ 1979; AMLACHER and RUDOLPH 1981; AMLACHER and R1JDOLPH 1982). The following test arrangement was employed: six suckling growing CBA mice 10-12 days old (5--6 g of body weight) of the same litter were used in each test experiment. 0.1 ml of the dissolved test chemical in a concentration of 30 per cent of the 21 hours DL50 was

193

applied by i.p. injection to 3 animals (test), whereas 3 mice were treated with the solvent only (control). 16 to 21 hours later the animals were given a single i.p. injection of 3H-thymidine (2 p,Cijg body weight) and 50 min afterwards they were sacrificed for autopsy and preparation of autoradiographs. Autoradiographs of slices of renal tubular and of liver epithelium were examined after a 4-day exposition by visual silver grain counting (50 nuclei per animal organ, that means 150 nuclei in each test and the same number in the corresponding control). We could demonstrate that well defined carcinogens of different chemical classes partially inhibit the nuclear incorporation of 3H-thymidine in a significant manner over a time period longer than 24 hours, causing a decrease in the mean of silver grain number over the marked nuclei. Noncarcinogenic compounds did not show this effect. The DNA synthesis of individual nuclei is not affected by the circadian rhythm (PILGRIM and MAURER 1965) opposite to the total num ber of nuclei in S phase (labeling index, "whole DNA synthesis") which depends also on growth stimulating agents. The uniformity of mean nuclear silver grain density persists continuously as well as both the 24 h time period and the time of entire S phase length. The suppressive effect of carcinogens on individual nuclear DNA synthesis as revealed by the decrease of silver grain number could serve as a criterion for suspicion of DNA damaging activity and therefore of possible carcinogenic action. It must be emphasized that the suppression of the so-called "whole DNA" synthesis, that means the suppression of the total of number of nuclei in S phase (autoradiographically demonstrable as labeling index) is not identical with the phenomenon of the individual suppression of nuclear DNA synthesis as was pointed out in our earlier published investigations (AMLACHER, DANZ, STILLER and RUDOLPH 1977: AMLACH ER und RUDOLPH 1978; AMLACHER und RuDOLPH 1981). Three of 53 tested well defined carcinogens were found to react as false negatives, that means the accuracy of the test (level of investigation until Jan. 1982) amounts to 94.5 %. Due to the fact, that all 26 tested non-carcinogenic compounds did not show a suppressive effect on nuclear individual DNA synthesis (not only in the kidney but also in the liver nuclei) the accuracy of the test in this ease amounts to 100 %) by missing of false positives (level of investigation until Jl1n.1982). 26 of new, synthesized industrial chemical compounds reacted in 10 eases with a signifieant nuclear DNA synthesis suppression (level of investigation until Jan. 1982). Under the standardized dosage conditions (30 % of the DL 50) the earcinogenic compounds could react by suppressing of both renal and hepatic nuclear DNA synthesis or by suppressing only the renal, respectively only the hepatic nuclear DNA synthesis. At time we cannot give an explanation of the biological basie events and mechanisms implicate in the phenomenon of individual nuclear DNA synthesis suppression by carcinogenic compounds in our assay. It is imaginable to interpret this effect at the molecular level as a damage of DNA synthesizing replicons, leading probably to later mutative changes. The postulated DNA damage persists over a time longer than 24 hours (AMLACHER and BOLCK 1977). Previously published results (AMLACHER, Dxxz, STILLER and RL'DOLPH 1977) revealed that minimal dosages (Lf) 0;, of the DL 50 ) of 9.1O-dimethyl-1,2-benzanthracene, respectively diethylnitrosamine given consecutively over 6 days, caused a marked suppressive effect on individual nuclear DN A synthesis. This was demonstrable in rats by 2/ 3 hepateetomy 10 days after the last application of the above mentioned low dosages and by silver grain counting over the nuclei of liver epithelia. in autoradiographic speeimens prepared 24 and 48 hours after hepatectomy. A damage of DNA synthesis regulating nuclear membrane factors is likewise conceivable. In an adequate test arrangement for inhalation the TSS can be used also to prove gaseous substances. The expeneses (photographie emulsion, radionuclides, test r.nimzls, foods) for testing one ehemical compound by the TSS remounts in the German Democratic Republic to 160 Marks (currency of the GDR). In this calculation the salaries are disregarded. A cautious estimation of possible expeneses in U.S. eurreney in the U.S.A. disregarding salaries amounts at present probablv to 10--20 1'.S. dollars for testing one chemical compound.

194

Discussion In the synop t ic table 2 so me results w er e compiled obtained with the five shor t term sc r ee ning assays di scussed b y us in th is work. It is self-ev ide n t that it is not po ssible to co m pare a ll the hitherto tested che m ica l co mp ou n ds be cau se mo st of them were tested onl y in th e AMES assay a n d not in eac h of th e a bo ve di scussed screening t ests. F or that reason w e have Tab le 2. Synoptic presentation of 40 carcinogenic and non- car cinogeni c compounds and their id entificati on by testing in the TSS, B:lIT, CTT, DCBA and USDT .

Chemical

Chemi cal compound 2-Acetylaminofluorene Acridine organe Actin om ycin D Aflatoxin Bi Alcohol, ethyl 4-Aminoazobenzene o-Aminoazotoluene 4-Ami nobiphenyl 1-Aminon aphthaline 2-Aminonaphthaline Aniline Ant hrace ne Arseni c Benzid in e (4,4-Diaminobiph en yl) 3,4-Benzpyrene Butansul tone Car bon tetraehloride Chro me VI D. D.T. (Dichlorodiphenyltri ehl orethan e) 4-Dieth yl-amino-azobenz ene Diethvln itro samine 4-Dinlet hy l-a mino-azobenzene 1,12-Dimethylbenz[aJan thracene Dim ethylformamide Dim ethylnitrosamine Dimethylsnlfate Dim ethylsulfoxide Lead acet at e 20-Met hylchola nt hrene Methylmethansulfonate N-nitroso-di-n-but ylamine N-m ethyl-N-nitro- Nnit rosoguanidine N-nitroso-N-met hylur ea N-nitroso-morpholine Ph enobarbital-Na 1,3 Propa ne sulfone Toluene 0- Toluidine Ure t hane, et hy l Ph enacet ine

class

genicity (Lit .)

Ca rcino -

TSS

AA PAll WrA

+

+ +

:11 AA AA AA AA

0

AA AA PAH

+

FTA

JI

AA

PAf!

+

+

+

+ +

+ (+) (+) (+) --I-

-+ +

+ +

+

ig.O

+ +

0

+ + -+

+

+

+ 0 +

0

ip.

DCBA

nt 0

+ +

nt

m IT CTT

+ + +

+

0 0 0

+ + +

+

nt nt

nt 0

nt

nt

+

nt nt 0 nt

nt

+

+

+

0 0 nt

+

-+ +

...L-

)l

+ +

+ + +

0 nt 0

nt nt 0

AA

0

0

0

nt

NA

-t-

--I-

+

+

0

0 nt

+

+

nt --II

+ +

+

+

+

+

+ +

0

0

0

nt

0

0

nt 0 0

nt nt nt

0

M M lVlI PAll EEC

NA NA

NA NA J)

EEC AA AA EEC

n

+ + + + + + + + + 0 + + I

T

+ +

+ + + + +

+ + + + 0 + + +

+

nt nt nt nt

+

0

+ +

+

nt

1'1

NA

+

nt nt nt

PA ll

-+-

+

nt nt nt

+

+ +

+ -+

AA

+

nt nt

nt nt 0 nt nt

+

E EC AU :l'I!

0

+

US DT

+

+ + + + + + 0 +

+ +

nt nt

+

+ + +

nt

+

nt

+ +

nt nt 0

nt nt

0 0

0 nt

nt nt

0

nt

0

-+

+

+

0

nt

+ +

nt 0 nt

+ + + + +

nt nt nt nt

+ +

nt

Denot ati ons: + = positive rea ct ion , 0 = negati ve reaction, nt = not test ed , ± uncertain result , (+) = pr oblemati c carcinogen icity, AA = ary l arnines and related compoun ds, AH = a lkyl halides, D = drugs, E EC = este rs, epox ides , ear ba mat es, F TA = fun gal t oxi ns, antibiotics, .M = miscellaneous, MI = met allic ions , NA = nit rnsa mines, PAH = polycyclic aro ma tic hydroc arbons, i.p, = intrap erit oneal inj ., i.g. = stomach intubati on

195

selected from the literature 40 frequently tested compounds of different chemical classes which were tested for the most part in th e five screening assays in discussion. Excluding l-aminonaphthaline, aniline and anthracene whose carcinogenicity is doubtful we see, that th e BMT (AMES) identifies 25 of 34, the CTT (STYLES) 18 of 20, the DCBA (Ku BISNKI ) 19 of 20, the USDT 17 of 18 and th e TSS (AMLACHER) 35 of 36 compounds in a correct manner. Co mparing th e above mentioned individual accuracies of th e five tests represented closely in thi s work it is obvious th at th e DCBA and the TSS seem to be th e most effective short-term assays. Regarding the relati ve simplicity and cheapness of th e TSS and with respect to th e fact th at th e mammalian organism can be used in its entirety without the necessity of an employment of a microsomal meta bolic aid fraction, it is evident th at thi s test arrangem ent offers manifold advantages. Alt hough t he fundam ental biological events controlling th e individual nuclear DNA suppr ession by eareinogens in th e TSS have not yet been elucidated at present , this eircumstanee seems to he without importance in th e practical employment of such a screening test as seen also in th e DeB assay. Contrarily to the DCBA and th e TSS, in th e BMT, the CTT and th e [SDT th e fundamental biological mechanisms are well known, however th e laboratory and apparativ e expenses reaeh a considerable level. This last statement applies also to the DeBA . The recent critical consideration s by RINKUS and LEGATOR (1979) referring to the BMT of AMES newly accentuate its partied imperfection which consists in the necessity of the use of different bacterial tester strains (cp. also McMAHOK et al.1979) and in the evident difficulti es by the constraint to use a metabolism activating microsomal fraction (S 9-mix). Difficulties were also manifest in testing volatil e, bactericid al and cross-link chemicals as point ed out by RINKUS and LECL\TOR ( I Hi H) in their thorough and detailed discussion. Recentl y, at the LOth Annual )Jeeting of the Europ ean Environmenta l Mutagen Society (Athens, Sept. ]980) th ere was again a debate ou th e problems of fundamental microbiological aspects of th e AMES test. th e influence of growt h media on th e yield of mutant colonies, th e effects of sponta neous mutati ons on th e sensit ivity of th e test, th e cell problems and th e influence of th e number of bacter ia on t he number of revert ant s etc. A collaborat ive study of the Alli ES assay between 19 Eur opean laboratorie s revealed considerable variations in t he test results ((;RAFE et al. 1H81; GRAFE 1981; GRAFE et al. 1981) leading to the following cunsequences: .I. " The amount and qualit y or t he S n preparation influenced th e results significantly . . . some of th e inter-lab orat orv variati ons is almost certainly due to differences in th e S 9 preparation used" . 2. "A second source of variati on appeared to be the number of cells routinel y plated; . . . Although only few laboratories reported the number of cells plated - and th e methods to determine this number differed considerably - it was clear that th e tests had been done with very different amount s of bacteria ... " 3. "As, in general ... a I fj . - 18 h culture was used for the tests, this indicates that overnight cultures contained very different amount s of bacteria in th e various laboratories. In addition, the bacteria may be in different growth phases or physiological conditions". 4. " Other sources of variati on include the type and amount of solvent used (because of their antibacterial activitv and intcrferenec with the activity of the enzymes in the S 9 mix), th e numb er of plate s user! for each concentration, and th e meth od for counting the numb er of colonies on the plat es ... .. GRAFE et al. (1981): " With t his additional information we can confirm that only with a different design of experiments it is possible to study th e reproductibilit y of the results in th e plat e incorporation assay" . GRAFE (1981): " If one searches th e lb lES t est literature for its microbiological basis, one is without success. Although one finds the requirement - repeated from one publication to another - that th e test should be carried out with more than 108 bacteria for plat e . .. no subst ant iation can

196

be found" .... "Out of 4 different phases in a statistical culture of bacteria only a limited section of the growth eurve is suitable for the AMES test". Many other aspects are also diseussed and criticized by GRAFE (1981). Additionally it must be emphasized that in a recent study PEAK et al. (1982) demonstrated that liver-microsome S 9 enzyme increases the spontaneous background mutation frequency in the AMES test system in the absence of any added mutagen. The spontaneous revertant number was by 30 with a high significance less than 0.01 at all 3 employed eoncentrations. The cell transformation test (CTT) is likewise an isi vitro assay and is also dependent on S 9-mix to activate seeondary carcinogens to their proximate and ultimate acting form. Its advantage consists in the employment of mammalian eell cultures allied with the possibility of additional eytological observations. In the Central Toxicology Laboratory of the Imperial Chemical Industries (Cheshire, England) the test is performed conrurrent with the AMES test (PURCHASE et al. 1978; STYLES 1977, 1978; ANDERSON 197\)). HUBERMAN et al. (1976) suggested that the ratio between transformation and mutagenesis for Ouabain resistance is about 20 : 1 after treatment with benzo(a)pyrene, that means anyone of 20 genes may be involved in transformation as opposed to 1 for mutation. Following A;-'DERSON (1979): "If transformation is therefore, a genetically based system, it may be useful for preliminary genetic monitoring. We have found it to be equally as predictive for carcinogenicity as the AMES test and by interference, therefore, it is equally predictive for mutagenicity ... The test is not as rapid as the AMES test ... ", The Unscheduled DNA test (P8DT) is based on well established cell biological knowledge and can be performed according to the statements of MARTIN et al. (1978) also by use of liqiud scintillation counting. However with this assay at present a too low number of chemical compounds was tested. MARTIN et al. (1978) accentuate that the U8DT opposite to the AMES assay indicates a smaller sensitivity in testing chemical compounds. Although the DNA cell binding assay (DCBA) seems to be an exclusively empirical screening test, nevertheless its accuracy is surprising in testing a greater number of chemical compounds and carcinogenic substances. Unfortunately the apparative equipment is very expensive and therefore its extensive employment is restricted. The dependence on pH and the unability to test gaseous compounds also render its use more difficult. Coincidentally the DCBA needs likewise the metabolic aid of a microsomal fraction (8 9-mix) to transform secondary carcinogens in their ultimate reactive form. Finally it remains to evaluate the thymidine incorporation inhibiting screening system ('1'88). At first sight the employed autoradiographical technique seems to be difficult. It must be admitted that the need for a quantitative autoradiographical evaluation by visual silver grain counting seems to be of little elegance and quickness in comparison with a photometrical analysis. However by comparing the time spent for counting of both, the visual silver grain evaluation is of greater rapidness (on an average of five seconds per one nucleus). Depending on the level of labeling index, for the analysis of 300 renal nuclei corresponding to one tested compound approximately 2--3 hours are wanted. If an additional evaluation of liver nuclei is needed, the total time of counting amounts to 6 hours. Taking into account a time space of 5 hours to test one chemical compound, then one worker is able to analyse on an average 200 chemicals in the course of one year. . The equipment for a T88-laboratory is very inexpensive. To test 600 chemicals per annum 3 workers and 3 microscopes are needed. Additionally except a scientific group leader 2 workers are needful for histological and autoradiographical working and one breeder for the test animals (mice). For breeding 3600 baby mice to test 600 chemical compounds all the year round, only 170 female and 36 male mice are necessary. Finally we can conclude that empirical short term tests like the DCBA and the T88 were of the same or even greater effectiveness and accuracy as seen by comparison with "classical" short term assays for instance the AMES assay or the Celltransformation test. 197

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