Heinz Loehrke

Diaplacental carcinogenesis: Initiation with the carcinogens dimethylbenzanthracene (DMBA) and urethane during fetal life and postnatal promotion with the phorbol ester TPA in a modified 2-stage Berenblum/Mottram experiment

Diaplacental initiation with the carcinogens DMBA or urethane followed by topical treatment of mice of F-1 generation with the tumor promotor TPA led to the formation of benign and malignant tumors on the back skin and also in various internal organs. (This system constitutes a modified 2-stage experiment based on the early schemes of Berenblum and Mottram.) Application of either the carcinogens or the tumor promoter alone did not lead to the formation of tumors within one year. The highest skin papilloma yield was obtained with mice initiated with DMBA from the 16–19th day of fetal life. The highest total tumor yield was obtained after initiation from days 18–21st. The combination urethane/TPA also promoted the formation of tumors of the skin and other organs. The significance of this modified prenatal-postnatal initiation/promotion scheme in human pathology is discussed.

Two-stage skin carcinogenesis by systemic initiation of pregnant mice with 7,12-dimethylbenz(a)anthracene during gestation days 6–20 and postnatal promotion of the F1-generation with the phorbol ester 12-tetradecanoylphorbol-13-acetate

SummaryThe DMBA-TPA-mediated two-stage skin carcinogenesis experiment was modified in that pregnant mice were systemically treated once during pregnancy with the carcinogen. Intragastric application times were fetal days 6, 8, and 10–20. A control group of pregnant mice received repeated doses (from days 8–20) of sesame oil, which was used as a solvent for DMBA.At the age of 12 weeks, the offspring of the control group were divided into two groups, one of which was left completely untreated, the other received TPA applications over 26 weeks.The 12-week old F 1-progeny of each transplacentally initiated group was also divided into subgroups, which either received no further treatment (subgroups A) or were promoted with TPA (subgroups B). Neither the F 1-animals of the two control groups nor that of the transplacentally initiated but postnatally not promoted subgroups 6A–20A developed skin tumors. The same holds true for the TPA-promoted offsprings of mother animals which had received DMBA at days 6 and 8 of gestation.Skin tumor development after TPA promotion was first observed in animals of subgroup 10B. Thereafter, tumor rates and tumor yields increased and latency periods decreased progressively in the B-subgroups with the postponenment of initiation to later fetal periods. Day 19 of prenatal development proved to be the most sensitive period to transplacental initiation, whereas initiation at day 20 led to a significant decrease in tumor rate and yield.The capability to initiate skin tumors and the extent of initiation can be correlated to both the organogenesis of the epidermis and its proliferative rate in utero.

Positive two-stage carcinogenesis in female sprague-dawley rats using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-o-tetradecanoylphorbol-13-acetate (TPA) as promotor

In contrast to a previous report by Shubik, the validity of the 2-stage skin carcinogenesis experiment was demonstrated in the rat. The modified experiment was carried out in female Sprague-Dawley rats using intragastrically administered DMBA as a carcinogen and the topically applied phorbol ester TPA as a promoter. Seven groups of animals were used. Two groups were treated with TPA only, two groups were initiated only with DMBA, two further groups were both initiated and promoted, and one group served as a control. Each of the initiated/promoted groups or only initiated or promoted groups contained one sub-group in which the animals had been bilaterally ovarectomized prior to the experiment. Hyperplasia of the dorsal epidermis occurred only in the promoted and in the initiated/promoted groups. Tumors of the back skin were observed exclusively after initiation/promotion. Ovarectomy — leading to a prolonged survival time of the animals — seems to be crucial for the manifestation of malignant skin tumors. Initiation/promotion also gives rise to tumors of the forestomach, the small intestine, the liver and the colon. Tumors in other organs (especially in the mammary gland and the Zymbal gland) were also be observed after initiation alone.

Transmaternal variation of the berenblum experiment with NMRI-mice

DMBA which is applied transmaternally via the mothers milk can initiate tumour cells in the F-1 generation. Subsequent application of the tumour promoter TPA to the back skin of the young animals induced the formation of skin papillomas and carcinomas. Animals treated according to this scheme also developed malignant neoplasms in the other organs. Control animals treated with DMBA only, rarely developed tumours, whereas treatment with TPA alone had no effect. This variation of the Berenblum-experiment suggests that the transfer of carcinogens in human milk should be considered in addition to transplacental carcinogen transfer as a potential hazard to the developing human infant.

Long term tumorigenicity of a single application of indomethacin or Amuno® in adolescent and in adult male Sprague Dawley rats

200 adolescent (Group I) and 200 adult male Sprague Dawley rats (Group II) were divided into 4 subgroups of 50 animals each. Animals were treated on the 29th (Group I) or 98th day of life (Group II) either with acetone or Amuno carrier in acetone or Amuno on acetone (2.5 mg indomethacin/100 g animal weight in acetone) or with the pure substance indomethacin 2.5 mg/100 g of animal weight in acetone, giving a single application on the shaved dorsal skin. Subsequently the animals remained under observation until their deaths, followed by autopsy and histopathologic examination of several organs. The rats treated with Amuno of indomethacin in the adolescent stage showed lower body weights and a shorter total survival time. The adolescent animals treated with Amuno or indomethacin showed a significantly higher rate of interstitial testicular tumors of the Leydig tumor type, adenomas and adenocarcinomas of the small and large intestine as well as hepatocellular tumors. The total number of neoplasias was higher in the animals treated with Amuno or indomethacin compared to those treated with acetone or carrier with acetone. Application of Amuno or indomethacin also resulted in a higher rate of hyperplasias (sole of foot, lymph nodes, prostate). The impairment of the synthesis of prostaglandins caused by indomethacin apparently results in starting complex pathomechanisms which have effects until the late death of the animals. Adolescent animals were affected more frequently by the application of indomethacin as were already adult animals.

Effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and its nonpromoting analogue 4-O-methyl-TPA on dorsal dermal melanocytes of the Syrian golden hamster (Mesocricetus auratus)

SummaryThe effect of the tumor promoter TPA and its inactive structural analogue 4-O-methyl-TPA on the induction of dorsal skin melanosis in the normal Syrian golden hamster and on the promotion of melanomas in DMBA-initiated animals was investigated. Both phenomena were observed in TPA-treated hamsters but could not be detected after exposure of animals to 4-O-methyl-TPA. In contrast to results obtained with a variety of other laboratory animals, neither TPA nor 4-O-methyl-TPA were able to induce epidermal hyperplasia of hamster dorsal skin.