Mouse Skin Papillomas Research Articles

Polyclonal origin of mouse skin papillomas.

We show that, from the earliest morphologically recognisable stages of development, mouse skin papillomas induced by a chemical initiation-promotion regime are polyclonal. We have demonstrated polyclonality directly by immunohistochemical staining of the mosaic cell populations in embryo aggregation chimaeras, a method which removes some of the uncertainties of previous conclusions based on analysis using electrophoretic polymorphisms. The findings may imply that initiation within a single cell and promotion of its clonal descendents is not a sufficient explanation for the origin of these tumours and that interaction between cells of more than one clone is involved.

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]pyrenes: the very potent environmental carcinogen dibenzo[a, l]pyrene

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with several dibenzo[a]-pyrenes (DBPs). SENCAR mice were initiated with DB[a, e]P, DB[a, h]P, DB[a, i]P, DB[a, l]P and anthanthrene, and promoted with tetradecanoyl-phorbol acetate. The same compounds were tested by intramammillary injection in female Sprague-Dawley rats. Anthanthrene was inactive in both mouse skin and rat mammary gland. DB[a, e]P was a very weak tumor-initiator in mouse skin and was inactive in rat mammary gland. DB[a, h]P induced twice as many papillomas in mouse skin as DB[a, i]P, although both compounds exhibited similar tumor latencies and percentages of tumor-bearing mice. These two compounds induced similar numbers of mammary tumors, but treatment of the rats with DB[a, i]P resulted in a significantly larger number of adenocarcinomas. DB[a, l]P was toxic to both the mice and rats. Treatment of mouse skin with this compound led to an erythema, which delayed the beginning of promotion until the 3rd week after initiation. Despite this delay, papillomas began appearing 5 weeks after initiation with DB[a, l]P and the number of tumors increased rapidly. The compound was so toxic in the rats that half of the animals died in the first 9 weeks and the remaining animals were sacrificed after 15 weeks. Nonetheless, DB[a, l]P was the strongest carcinogen tested, inducing seven tumors per rat within 10 weeks. These results demonstrate that DB[a, l]P, which is present in tobacco smoke, is an extremely potent carcinogenic aromatic hydrocarbon. Furthermore, some of these compounds can serve as useful models for elucidating their mechanisms of activation.

Sequential trisomization of chromosomes 6 and 7 in mouse skin premalignant lesions

Using a direct cytogenetic technique, we identified a nonrandom trisomy of chromosome 6 in 12 of 12 aneuploid mouse skin papillomas and in 10 of 11 squamous cell carcinomas induced by chemical carcinogenesis. The second most common abnormality observed was trisomy of chromosome 7 found in most dysplastic papillomas and in 10 of 11 carcinomas. The two trisomies were the only abnormalities found in all aneuploid papillomas and in several carcinomas. Mutation at codon 61 of the Ha-ras gene, which resides on chromosome 7, was also a common feature of the tumors sampled. Extensive homology exists between mouse chromosome 6 and human chromosome 7, the trisomy of which was recently suggested as a primary cytogenetic event in several human epithelial cancers. We propose a multistep model of tumor progression in which a sequence of specific nonrandom chromosomal abnormalities appear to be required for malignant transformation.

N-Nitrosocimetidine as a modifier of chemically-initiated tumors in mice

N-Nitrosocimetidine (NCM) is a nitrosation product of cimetidine, a commonly-prescribed pharmaceutical agent. In spite of its known genotoxicity, NCM has failed to cause tumors in assays with rats and mice, but has given indications of enhancing or suppressive effects on tumor development. This possiblity was tested by administering NCM topically to the skin or in the drinking water to mice in which tumors had been initiated by treatment with chemical carcinogens. Sencar mouse skin papillomas initiated by 7,12-dimethylbenzanthracene (DMBA) and promoted by 12-O-decanoylphorbol-13-acetate (TPA), progressed more rapidly to carcinoma on mice given treatment during stage 3 (after TPA) with NCM (1 mg/week) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG, 120 g/week) than on stage 3 acetone controls. Oral NCM (1 g/l drinking water) did not have this effect but rather suppressed development of keratoacanthomas, as did stage 3 MNNG or TPA. Primary lung tumors initiated in BALB/c mice by i.p. injection of urethane; and tumors of forestomach, lung, mammary, lymphoid and skin tissues caused in ( C57BL 6 × DBA 2 )F 1 mice by oral DMBA were not markedly affected by NCM given in drinking water (1000–1800 ppm) until 14–16 months of age. These results confirm NCM's general lack of activity as an in vivo toxicant, but show that under certain circumstances it may enhance or suppress tumor development

Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.

Expression pattern of a gene for a secreted metalloproteinase during late stages of tumor progression.

We have reported that transin RNA, a 1.9-kb RNA coding for a novel, secreted proteinase, was overexpressed during the progression of benign mouse skin papillomas to malignant squamous cell carcinomas (SCCs) induced by a two-stage protocol (Proc Natl Acad Sci USA 83:9413, 1986). Recently a high degree of similarity has been demonstrated between rabbit stromelysin, a secreted metalloproteinase that degrades proteoglycans found in the basement membrane and the amino acid sequence predicted in rat transin cDNA. DNA sequencing of a mouse cDNA isolated from an SCC (initiated by 7,12-dimethylbenz[a]anthracene [DMBA] and promoted by 12-O-tetradecanoylphorbol-13-acetate [TPA]) showed greater than 85% nucleotide similarity and 90% amino acid similarity to the rat transin-1 cDNA nucleotide and predicted amino acid sequences. Using this mouse transin cDNA clone as a probe (labeled with 32P) we found enhanced levels of transin mRNA transcripts in SCCs induced by a protocol giving rise to metastatic tumors (repeated N-methyl-N-nitroso-N'-nitroguanidine [MNNG] treatments) compared with the level found in SCCs induced by a protocol that had a lower probability of giving rise to metastatic tumors (MNNG initiation followed by TPA promotion). A study of primary SCCs and metastatic lesions induced by repeated benzo[a]pyrene treatment showed that the levels of transin mRNA transcripts were reduced in the metastatic lesions in comparison to the primary tumors. Southern analysis of the DNA isolated from epidermis, papillomas, and SCCs indicated that neither transin gene amplification nor rearrangement accounted for increased levels of the transin mRNA transcripts. These data suggest a role for enhanced levels of transin production in the invasion and metastasis of chemically induced SCCs.

Effect of Retinoic Acid on Cornified Envelope Formation: Difference Between Spontaneous Envelope Formation In Vivo or In Vitro and Expression of Envelope Competence

A large number of cross-linked envelopes form spontaneously when cell lines derived from chemically induced mouse skin papillomas are cultured in medium containing 1.2 mM calcium. This phenomenon is associated with high activity of the cross-linking enzyme, epidermal transglutaminase (TGase). The influence of retinoic acid (RA) on envelope formation was studied in detail in a papilloma cell line, PE. Retinoic acid (3 microM) completely blocked cornified envelope (CE) production but reduced TGase activity only 50%. A rabbit antiserum was produced against sonicated CEs isolated from newborn mouse skin. On Western blots of epidermal extracts, diffuse staining was observed for particulate proteins of suprabasal, but not basal, cells and similar immunoreactive material was absent from the cytosolic fraction of both cell layers. The antibody also recognized particulate proteins from PE cells induced to differentiate by calcium, but not from cells grown in the presence of high calcium and RA. The antiserum appears to recognize partially cross-linked CE precursor proteins judging by the diffuse staining, the molecular weight range of the proteins stained, and their origin in the particulate cellular fraction. Cross-linked envelopes could be induced in RA-treated PE cells by permeabilization with 0.75 M NaCl or 50 micrograms/ml A23187. However, this treatment failed to cause the appearance of proteins recognized by the antiserum. Preincubation of the antiserum with purified fragments of CEs from newborn mouse epidermis, but not with cross-linked envelopes from permeabilized, RA-treated PE cells, removed immunoreactivity. These results indicate that the cross-linked envelopes formed in RA-treated cells after permeabilization lack a set of proteins contained in CEs from stratum corneum and may even be composed of different proteins. Retinoic acid appears to prevent CE formation in part by inhibiting activation of epidermal TGase but in addition by influencing the synthesis of precursor proteins.

Progressive dysplasia and aneuploidy are hallmarks of mouse skin papillomas: relevance to malignancy.

We report a systematic histopathologic study of papillomas at different times during promotion, correlating the results with those from cytogenetic analysis of the same tumors. Papillomas were induced in SENCAR mice by two-stage carcinogenesis (7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate). Individual tumors were randomly sampled at different times during promotion, and histopathologic and cytogenetic studies were carried out on every tumor. Early during promotion (10 weeks), most papillomas were well-differentiated hyperplastic lesions with mild or no cellular atypia. No tumors showed severe dysplastic changes. By 20 weeks of promotion, a dramatic drop had occurred in the number of lesions with no dysplasia. Most of the tumors presented moderate dysplasia, and some already showed severe dysplastic changes. At later stages (30-40 weeks), most of the papillomas were classified as moderately or severely dysplastic papillomas, and several were considered to be intrapapillomatous carcinomas. This histopathologic evaluation was supported by nuclear measurements performed on papillomas at different time points. Chromosomal abnormalities followed a similar trend. Papillomas seem to start as diploid lesions, but between 10 and 20 weeks of promotion, hyperdiploid cells can be observed in almost every tumor. In some cases the stem line was taken over by aneuploid clones. At 40 weeks of promotion, all papillomas were aneuploid, most of them with hyperdiploid stem lines. A positive correlation was found between the histological and cytogenetic studies, with the most aggressive and atypical tumors being the more aneuploid. These results support the idea that most, if not all, papillomas are truly premalignant lesions in different stages of the potential progression toward malignancy. Chromosomal abnormalities might play an important role in the sequence of events leading to malignancy.

Studies of skin tumorigenesis in PGK mosaic mice: many promoter-independent papillomas and carcinomas do not develop from pre-existing promoter-dependent papillomas.

The induction of mouse skin papillomas in two stages by initiation-promotion protocols has been studied extensively. On the basis of kinetics of induction and morphological observations of apparent progression to carcinomas, it has been argued that the papillomas sequentially advance to carcinomas through promoter-dependent and promoter-independent premalignant stages. Although this hypothesis has been widely accepted, the step-wise development of the same cell population from papilloma to carcinoma has not been established at the cellular level. We show here that some carcinomas which morphologically appear to result from progression of promoter-dependent papillomas do not develop from sequential progression of the same cell population as was found in the original papillomas. Other initiated cells also participate during the growth of these tumors and may replace the original papilloma cells. We also present evidence that some tumors develop directly as promoter-independent papillomas without going through an apparent promoter-dependent papilloma stage.

Sequential studies of skin tumorigenesis in PGK mosaic mice: the effect of repeated exposure to a carcinogen on regressed mouse skin papillomas.

Most mouse skin papillomas developing after 7,12-dimethylbenz[a]anthracene (DMBA) initiation followed by repeated 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion are promoter-dependent; termination of promotion results in their regression. Previous evidence, from mapping the locations of papillomas and using the X-chromosome-linked phosphoglycerate kinase cell markers, shows that regression of promoter-dependent papillomas is permanent. Exposure to another course of TPA promotion was found not to induce regeneration in the regressed papillomas. To determine whether repeated exposure to a carcinogen causes regeneration of regressed papillomas, the effects of weekly applications of low doses of DMBA were tested. The results reported here indicate that regressed promoter-dependent papillomas do not regenerate when exposed repeatedly to DMBA. However, the treatment with DMBA induced many new tumors most of which were promoter-independent papillomas or malignant carcinomas. These findings provide further support for the conclusion that termination of promotion leads to permanent regression of most promoter-dependent mouse skin papillomas.

Analysis of the rasH oncogene and its p21 product in chemically induced skin tumors and tumor-derived cell lines.

Mouse skin papillomas and squamous cell carcinomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with phorbol esters, such as 12-O-tetradecanoyl phorbol-13-acetate, frequently contain an activated Harvey ras gene. Six murine epidermal cells lines established from pooled skin papillomas previously tested negative in the NIH-3T3 assay, but have an altered differentiation program by a variety of criteria. The Harvey ras gene and its p21 protein product from these cell lines have been analyzed for alterations responsible for their altered growth and differentiation properties that were undetectable by 3T3 transfection assays. In comparison with primary papillomas and carcinomas, shown to have a point mutation in codon 61 of the Harvey ras gene, resulting in a p21 product with the diagnostic alteration in SDS-PAGE, the papilloma cell lines exhibited neither the codon 61 mutation, nor p21 product with altered migration in SDS-PAGE. These findings suggest that these papilloma cell lines contain a genetic lesion(s), other than Harvey ras activation, that may be responsible for their altered epithelial differentiation patterns and thus may serve as a useful model for identifying lesions involved in malignant conversion.

Mouse keratinocytes derived from initiated skin or papillomas are resistant to DNA strand breakage by benzoyl peroxide: a possible mechanism for tumor promotion mediated by benzoyl peroxide

Alkaline elution was used to examine DNA single-strand breaks in cultured normal and carcinogen-altered mouse keratinocytes exposed to 12-O-tetradecanoyl phorbol-13-acetate and benzoyl peroxide. Seven cell lines derived from carcinogen-induced mouse skin papillomas and three cell lines derived from N-methyl-N'-nitro-N-nitrosoguanidine-treated non-tumor bearing mouse skin were resistant to phorbol ester-mediated DNA strand breaks after 6 or 24 h. Normal keratinocytes sustained strand breaks after 24 h but not after 6 h. Benzoyl peroxide induced extensive strand breaks in normal keratinocytes at both 6 and 24 h, and this was associated with marked cytotoxicity. In contrast, 9 of 10 cell lines showed complete or partial resistance to strand breaks following benzoyl peroxide exposure. It is proposed that differential resistance to DNA strand breaks and cytotoxicity among normal and carcinogen-altered keratinocytes provides the biological basis for the promoting action of benzoyl peroxide. Furthermore, sublethal DNA damage in preneoplastic or neoplastic keratinocytes may account for the potency of benzoyl peroxide in causing malignant conversion.

Effect of application frequency on epidermal ornithine decarboxylase induction by chrysarobin in SENCAR mice

Ornithine decarboxylase (ODC EC 4.1.1.17) induction in mouse epidermis after single or multiple topical applications of chrysarobin differed from that following topical application of 12- O-tetradecanoylphorbol-13-acetate (TPA). Following a single application of 220 nmol chrysarobin, ODC activity began to rise at 24 h reaching a peak at 56 h and returned to normal after 96 h. When 5 separate applications of 220 nmol chrysarobin were applied in multiple application protocols, an alteration in the ODC induction response was observed. With a once per week or twice per week application protocol, ODC was elevated in a multiphasic manner giving multiple peaks of activity after the last application. Interestingly, the magnitude of ODC induction was greater using the once per week, compared to the twice per week, application protocol. Preliminary results indicate that a once per week application protocol is more effective than a twice per week protocol for promoting the development of skin papillomas in SENCAR mice. Thus, the magnitude of the induced ODC response with chrysarobin, although low compared to TPA, correlated with tumor promoting activity.

Transfection of the EJ rasHa gene into keratinocytes derived from carcinogen-induced mouse papillomas causes malignant progression.

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.

Transfection of the EJ rasHa Gene into Keratinocytes Derived from Carcinogen-Induced Mouse Papillomas Causes Malignant Progression

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.

Malignant progression of mouse skin papillomas treated with ethylnitrosourea, N-methyl- N′-nitro- N-nitrosoguanidine, or 12- O-tetradecanoylphorbol-13-acetate

Mouse skin tumors were induced by a single topical application of 7,12-dimethylbenzanthracene (DMBA), followed by biweekly promotion with 12- O-tetradecanoylphorbol-13-acetate (TPA). After 20 weeks of promotion, mice were treated twice weekly for 2 weeks with either ethylnitrosourea (ENU), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) or TPA. Thereafter all groups were treated biweekly with TPA. The ENU-treated group had a higher percentage of animals with carcinomas and developed 217% more cumulative carcinomas per group than TPA-treated controls. The percentage of mice with carcinomas and the cumulative number of carcinomas per group in MNNG-treated mice was higher than TPA-treated controls but was less than ENU-treated mice. The ratio of cumulative carcinomas to cumulative papillomas in ENU treated, MNNG-treated and TPA-treated mice was 16%, 9% and 6%, respectively. Histological examination of tumors remaining at the termination of the experiment revealed the presence of keratoacanthomas, some of which stained positive for γ-glutamyltransferase (GGT), in the ENU-treated and MNNG-treated, but not the TPA-treated groups. The fact that no new papillomas developed during the progression stage indicated that enhanced carcinogenesis resulted from the progression of pre-existing tumors. Enhanced progression of benign skin tumors in mice by only a few treatments of an agent may serve as a potential model for studies into the mechanisms and the inhibition of malignant progression. The model also allows for a comparison of the potency of agents in enhancing malignant progression.

A direct cytogenetic technique for mouse skin carcinomas and papillomas

A method was developed for obtaining direct chromosome preparations from SENCAR mouse skin tumors induced by chemical carcinogenesis protocols. Papillomas and squamous cell carcinomas were mechanically dispersed immediately after resection and were placed in a modified Hanks' solution with collagenase, trypsin, hyaluronidase, bovine albumin, and Colcemid. Total exposure to Colcemid did not exceed 1 hr. Metaphases were obtained in 100% of the analyzed specimens, allowing chromosome counting screening for double minutes and, in 50% of the cases, useful G-banded slides. The technique described has produced, for this type of tumor, a higher number of successful G-banded preparations than other previously reported methods for solid tumors. This procedure may be applicable for the study of human solid tumors that are histologically similar to our murine model, such as squamous cell carcinoma of cervix or lung.

Cultivation and characterization of cells derived from mouse skin papillomas induced by an initiation-promotion protocol.

Methods to culture cells from papillomas induced by an initiation-promotion protocol in SENCAR mice were developed, and the resultant cell lines have been characterized. Using Eagle's medium with 0.05 mM Ca2+ conditioned by dermal fibroblasts and supplemented with 1 ng/ml epidermal growth factor (EGF) in culture dishes coated with collagen and fibronectin, six cell lines (PA, PB, PC, PD, PE and PF) were established from separate pools of papillomas. When tested for tumorigenicity in nude mice by injection of a cell suspension or implantation of cells growing on a plastic liner, two of the lines (PC and PF) produced no tumors at any passage. In contrast, cells of the lines PA and PE produced highly differentiated squamous cell carcinomas from the earliest passage tested. The results with PB and PD were variable on tumorigenicity testing with some passages positive and others negative. When tested for responsiveness to Ca2+ (greater than 0.1 mM) as a differentiation stimulus, all lines responded. In the higher Ca2+ medium there was a 50-95% decrease in colony-forming efficiency, a slight decrease in [3H]thymidine incorporation (except for PA) and an increase in the number of cornified cells (except for early passage PF). Epidermal transglutaminase activity, a marker for terminal differentiation, was increased in the presence of medium with Ca2+ greater than 0.1 mM. However, unlike normal cells, only a fraction of the cells from each of the papilloma-derived cell lines terminally differentiated in response to Ca2+ while the remaining cells continued to proliferate, although at a slower rate. Responsiveness to phorbol ester tumor promoters was also examined in papilloma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment increased colony forming efficiency, DNA synthesis and colony size in all lines in medium with either 0.05 mM Ca2+ or 1.2 mM Ca2+. TPA treatment also increased ornithine decarboxylase activity in all lines, even at the higher Ca2+ concentration, although normal keratinocytes respond only when grown in medium with low Ca2+. TPA treatment caused only a slight increase in the number of cornified cells and no increase in epidermal transglutaminase activity in papilloma cells while it causes 10-fold or greater increases in these differentiation markers in normal keratinocytes. Thus papilloma cells appear to differ from normal keratinocytes in their ability to maintain a proliferating population under conditions favoring terminal differentiation, their consistent proliferative response to phorbol esters under these same conditions, and their reduced sensitivity to phorbol ester-induced terminal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Technical approach for cytogenetic studies of mouse skin papillomas and carcinomas : C.M. Aldaz, C.J. Conti, A.J.P. Klein-Szanto, T.J. Slaga. University of Texas System Cancer Center, Science Park-Research Division, Smithville, TX 78957.

Technical approach for cytogenetic studies of mouse skin papillomas and carcinomas : C.M. Aldaz, C.J. Conti, A.J.P. Klein-Szanto, T.J. Slaga. University of Texas System Cancer Center, Science Park-Research Division, Smithville, TX 78957.

The activities of 2,2',5,5'-tetrachlorobiphenyl, its 3,4-oxide metabolite, and 2,2',4,4'-tetrachlorobiphenyl in tumor induction and promotion assays.

Sensitive assays for the induction of lung adenomas in A/J mice or skin papillomas in SENCAR mice failed to show activity for either 2,2',5,5'-tetrachlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl 3,4-oxide. Injections of the 3,4-oxide into preweanling A/J mice caused considerable mortality, whereas the parent hydrocarbon did not. Both 2,2',5,5'- and 2,2',4,4'-tetrachlorobiphenyl showed promoting activity for hepatic gamma-glutamyl transpeptidase-positive foci initiated in rat liver by treatment with diethylnitrosamine. The promoting activity of 2,2',4,4'-tetrachlorobiphenyl was approximately 10-fold greater than that of the 2,2',5,5'-isomer.