Promotion of skin tumours by TPA in the progeny of mice exposed pre-natally to DMBA

Pregnant SHR mice were treated once with 7,12-dimethyl-benz[a]anthracene(DMBA) on day 17–19 of gestation, and F₁ and F₂ descendantsreceived multiple skin applications of 12-O-tetradecanoylphorbol-13-acetate(TPA) twice a week for 24 weeks beginning at 12 weeks of age.Post-natal promoter treatment resulted in a high incidence ofskin twmours in F₁ and F₂ mice (37.3 and 19.7%, respectively),whereas only 6.6% of control animals treated with TPA only developedskin tumours. DMBA was shown previously to be capable of initiating skin carcinogenesis transplacentally; however, ourresults on the second generation provide suggestive evidenceof hereditary transmission of at least part of the initiatingaction of this carcinogen.     

On the persistence of tumour initiation and the acceleration of tumour progression in mouse skin tumorigenesis

Evidence has been obtained for the reversibility of initiation of carcinogenesis as evoked by 100 μ 7,12-dimethylbenz (a)anthracene (DMBA) applied to the skin of Swiss mice. Mice exposed to twice-weekly applications of a phorbol ester, TPA, for 15 weeks developed multiple papillomas when treatment was started 3 weeks after tumour initiation with DMBA, but very few when the interval was 50 weeks. This finding is not necessarily at variance with the postulate of the irreversibility of formation of “latent tumour cells” by subcarcinogenic doses of DMBA. Intraperitoneal injections of urethane increased the risk of development of malignant skin tumours by mice bearing multiple papillomas as a result of previous treatment with 100 μMg DMBA and TPA as compared with intraperitoneal injections of distilled water. This finding may allow a more clear-cut experimental definition of the stages in the process of tumour progression in mouse skin tumorigenesis.     

Inactivity of fecapentaene-12 as a rodent carcinogen or tumor initiator

The possible carcinogenic activity of synthetic fecapentaene-12 (FP-12) was studied in several mammalian test systems: (a) for carcinogenicity by intrarectal instillation in male F344/NCr rats as well as by intrarectal and subcutaneous application in male B6C3F1 mice; (b) for initiation by skin painting in female SENCAR mice followed by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), with 7,12-dimethylbenz[a]anthracene (DMBA) followed by TPA as positive control; (c) in a rat subcutaneous granuloma pouch assay in which mutagenicity was measured by induction of 6-thioguanine (6-TG) resistance and carcinogenicity was determined by induction of subcutaneous tumors in the pouch. There was no significant increase in tumor incidence after 72-78 weeks in test (a), although 2 rats receiving FP-12 intrarectally developed colon polyps. FP-12 did not initiate any skin tumors in test (b), nor did it significantly convert DMBA-initiated papillomas into carcinomas when 8 of the positive control mice were given FP-12 weekly for 10 weeks after 10 weeks on the DMBA-TPA regimen. Although FP-12 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were comparably mutagenic in test (c), FP-12 induced no tumors after more than a year in 133 rats at risk while MNNG induced 7 tumors in 107 rats. These rodent assays provide no evidence that FP-12 is a strong carcinogen, although the possibility remains that it may possess weak carcinogenic activity not revealed by these experiments.     

Tumour initiatory activity of a herbicide diuron on mouse skin

In the present investigation, the tumour initiating activity of a herbicide diuron 3-(3,4 dichlorophenyl)-1,1 dimethyl urea has been observed following multiple topical applications at a dose of 250 mg/kg body weight in a standard two-stage initiation-promotion protocol on mouse skin for carcinogenicity testing. It was found that 9 applications of the herbicide given on the interscapular region in a thrice weekly schedule up to 3 weeks and followed, after 1 week, by the repeated 3 times per week application of a known skin tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), 5 micrograms dissolved in 100 microliters of acetone in the same initiated area, led to the development of benign skin tumours. However, a single dose of diuron, used for each application as above and followed by repeated TPA applications, with the same dose and painting schedule as in the case of multiple applications, failed to initiate tumour development.     

Diaplacental initiation of NMRI mice with 7,12-dimethylbenz[a]anthracene during gestation days 6--20 and postnatal treatment of the F1-generation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate: tumor incidence in organs other than the skin

The tumor spectrum and tumor incidence in organs other thanthe skin were investigated In the 12-O-tetradecanoylphorbol-13-acetate(TPA) treated F1-generation of 13 groups of NMRI mice whichhad been initiated by a single Intragastric dose of 7, 12-dimethylbenz[a]anthraceneduring days 6, 8, and 10–20 of pregnancy. Promotion bytopical application of TPA to the back skin was carried outtwice per week 12 weeks after birth over a period of 26 weeks.The forestomach epithelium represented the only organ in whichstatistically significant 2-stage carcinogenesis could be demonstrated.A promotion effect could be seen in tumors of the Harderlangland, of the liver of male animals and on the development ofboth benign and malignant tumors of the lung in both sexes.Promotion treatment therefore led to an activation of initiatedtumor cells in those organs in which a very sensitive, moreor less narrowly spaced oncogenic determination period exists.     

Carcinogenicity of DDT (dichlorodiphenyl trichloroethane) in pure inbred Swiss mice

Inbred Swiss mice were treated with technical DDT (1) orally with the diet or by intubation; (2) subcutaneously and (3) by skin painting. The total duration of the experiment was 80 weeks. There was no difference in body growth and mortality between the experimental and control groups. Toxic manifestations of DDT were observed in treated mice in the form of tremor, convulsions and corneal opacity usually after 40 weeks. Oral and subcutaneous DDT treatment resulted in a significant increase in the incidence of tumours mainly of lymphoid tissues, lung and liver. The highest tumour incidence was recorded in the group of mice receiving DDT by subcutaneous injections. Males and females were equally susceptible. No evidence of carcinogenicity was observed in the painted group.     

Sandalwood oil prevent skin tumour development in CD1 mice.

Sandalwood oil (SW oil) has been used for the treatment of inflammatory and eruptive skin diseases. In the present study, the chemopreventive effects of SW oil on 7,12-dimethylbenz(a)-anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate(TPA)-promoted skin tumour development and TPA-induced ornithine decarboxylase (ODC) activity in CD1 mice were investigated. Female CD1 mice (5-6 weeks old) were divided in different groups, having 30 mice in each group. One week after topical application of DMBA (200 nmole in 100 microl acetone) alone or with SW oil at different concentrations (100 microl, 1.25, 2.5, 3.75, 5% in acetone), at different times (0.5, 1, 2 h) before DMBA, the mice were treated topically with TPA (5 nmole in 100 microl acetone) alone or with SW oil at different concentrations (100 microl, 1.25, 2.5, 3.75, 5% in acetone) at different times (0.5, 1, 2 h) before TPA applications twice a week for 20 weeks. The mice were weighed and papillomas counted weekly. The results indicate that SW oil pre-treatment decreased the papilloma incidence and multiplicity in a concentration and time-dependent manner. The pre-treatment with 5% SW oil (100 microl) 1 h before DMBA and TPA treatments provided a maximum of 67% and 96% decrease in papilloma incidence and multiplicity, respectively, after 20 weeks of promotion. The mice pre-treated with SW oil at all concentrations and time period before TPA had significantly lower ODC activity than the group treated with TPA alone. The data suggest that SW oil could be an effective chemopreventive agent against chemically-induced skin cancer.     

Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin.

Protein A is an immunostimulating glycoprotein obtained from Staphylococcus aureus Cowan I. Its antitumour activity is proven in various tumour models. Its ability to provide protection against tumour initiation by the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA) has been investigated in the present study using a mouse skin model of two-stage carcinogenesis. Protein A was administered intraperitoneally (1 microgram/animal 20 g body wt.) twice a week for 2 weeks, prior to initiation by DMBA. The promotion was performed by twice weekly applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (3 or 5 micrograms/animal in 100 microliters acetone). Protein A provided significant protection to animals from DMBA-induced tumour initiation as was observed by the decrease in cumulative number of tumours, percent of animals developing tumours, number of tumours per animal and rate of tumour growth. Our data indicate that protein A has anticarcinogenic properties.     

Sensitivity of the skin of different mouse strains to the promoting effect of 12-0-tetradecanoyl-phorbol-13-acetate

The sensitivity of the skin of Swiss DBA/2 and C57B1/6 mice was compared in two-stage carcinogenicity experiments. Groups of 30 mice were treated once with 7,12-dimethylbenz(a)anthracene (DMBA) (50 micrograms/mouse) which was applied to the shaved dorsal skin as initiator and, starting one week later, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was applied thrice-weekly at doses of either 0.04, 0.16 or 0.64 micrograms/mouse to the areas of initiated skin. Other groups of mice were similarly treated with TPA (0.64 micrograms) alone. The times at which papillomas and carcinomas first appeared were recorded. Overall the results show, in terms of the numbers of animals with tumours, the total numbers of tumours produced and the numbers of malignant tumours formed all in dose-response relationship, that the Swiss mice were the most sensitive and the B57B1/6 mice the least sensitive to the tumour-promoting effects of TPA with the DBA/2 mice occupying and intermediate position. This relationship also held in the control groups that were treated with TPA alone.     

The effect of the irradiation of male mice on the sensitivity of 2 generations of progeny to the action of the skin tumor promoter 12-0-tetradecanoylphorbol-13-acetate

Progeny of male mice, either irradiated in a single dose of 4.2 Gy prior to mating or intact, were painted with either acetone or acetone solution of 6.15 micrograms 12-0-tetra-decanoylphorbol-13-acetate (TPA) twice a week for 24 weeks from the age of 4 months. The occurrence and number of skin papillomas were registered at 2 and 20 weeks after the final treatment. Painting with acetone was not followed by tumor growth in the progeny of either irradiated or intact mice. At 2 weeks after TPA treatment, the occurrence of skin papillomas in the male and female progeny of unirradiated mice was 21 and 37% whereas at 20 weeks-12 and 15%, respectively. The occurrence of skin papilloma in the irradiated mice progeny at 2 weeks was 75% males and 50% in females whereas at 20 weeks-68 and 43%, respectively. Some F1 progeny of the irradiated male mice were mated prior to TPA treatment, and F2 progeny were treated with either acetone or TPA. In the latter group, the occurrence of skin papilloma at 2 weeks following promotor treatment was 58% for males and 40% for females whereas at 20 weeks--53 and 36%, respectively. The analysis of skin papilloma occurrence by family showed that considering the papilloma-bearing father or mother relationship, the former factor appeared to be responsible for a stronger predisposition to this lesion development in F2 progeny. However, the number of papilloma--bearing animals was the greatest in families where both parents suffered the disease.     

Transplacental and transgeneration carcinogenic effect of 7,12-dimethylbenz[a]anthracene: relationship with ras oncogene activation.

Transgeneration transmission of the carcinogenic action of 7,12-dimethylbenz[a]anthracene (DMBA) was studied in two generations of mice using transplacental DMBA initiation followed by postnatal skin tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the first generation (F0) and only promotion in the second generation (F1). Local application of TPA resulted in increased skin tumor yield in both the in utero DMBA-exposed mice and their progeny (P = 0.0002 and P = 0.0941 respectively compared to control). Similarly, lung tumor incidence was increased in the two generations of mice (P less than 0.0001 and P = 0.0080 respectively). The results suggest transgeneration transfer of the effect of DMBA. A to T mutation at the second base of codon 61 of the Ha-ras oncogene was found in skin tumors of DMBA-exposed mice, but not in tumors induced by TPA without initiation. Analysis of Ki-ras codon 61 in seven lung tumors from DMBA-treated mice revealed three types of mutation: two cases with CA[C or G or T], one case with CCA and one case with CTA (the remaining cases having only the wild type). Six of these mice also had skin tumors, which contained A to T mutation at the second base of codon 61 of the Ha-ras gene in five cases. Thus mutations of different ras genes were found in skin and lung tumors from the same animals. In the progeny (F1) of DMBA-exposed F0 mice, only skin tumor samples were available for oncogene analysis and none contained the Ha-ras mutation. The results confirm our previous finding that initiation of skin and lung tumorigenesis can be transmitted transgenerationally. On the other hand, our data from a limited number of skin tumors suggests that ras gene mutation may not be critically involved in this transmission.     

Possible role of genetic predisposition in multigeneration carcinogenesis

A peculiar phenomenon in experimental transplacental carcinogenesis is that in certain animal species or strains, and with certain types of carcinogens, a tumorigenic risk is observed not only in the F1 generation but also in subsequent F2 and even F3 generations when only the P generation has been exposed to a carcinogen. Additionally, in many cases, specific types of organs tend to be involved. For example, lung tumours are most common in P, F1 and F2 generations of Swiss, ICR, MA and CD-1 mice after exposure of the pregnant P generation to 7,12-dimethylbenz[a]anthracene (DMBA), urethane, DMBA and diethylstilboestrol, respectively. In such mice, the mammary glands, lymphatic tissues and ovaries are also frequently involved. Recently, two-stage tumorigenesis in skin with a phorbol acetate as promoter was transmitted to F2 descendants born of F1 SHR mice exposed transplacentally to DMBA. In WKA, BD IV and BD VI rats, nervous tissues seem to be prone to tumour development in F2 and/or F3 descendants born of F1 exposed transplacentally to N-methyl-N-nitrosourethane, N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea (ENU), respectively. In F344 strain rats, however, two-stage genesis of preneoplastic foci in the liver with 2-acetylaminofluorene (2-AAF) as the promoter failed to be transmitted to F2 and F3 generations from the F1 generation, which developed the preneoplastic hepatocellular foci after prenatal exposure to ENU followed by postnatal promotion by 2-AAF. Our own recent experiments with Syrian hamsters, in which multigeneration transmission of organ-specific (tracheal epithelium) tumorigenicity of N-nitrosodiethylamine was investigated, have also failed to show this phenomenon in F2 and F3 descendants. However, various field studies on pedigrees with frequently affected siblings indicate the existence of a heritable predisposition to tumour development over more than two generations. The organs involved include ovaries, mammary glands, stomach, lympho- and myelogenic systems, skin and nervous system. Interestingly, little or no such evidence has been found in the liver, respiratory tract (except for the nasopharyngeal region) or urinary tract. In the light of these experimental and human observations, the authors are currently inclined to hypothesize a heritable predisposition to tumour development in organs specific to animal species and strains, or certain families in human cases, for explaining the mechanisms underlying multigeneration transmission of chemical carcinogenicity.     

Effects of dose and duration of treatment with the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate on mouse skin carcinogenesis

The effects of dose and duration of treatment with the potenttumor-promoting agent 12-O-tetra-decanoylphorbol-13-acetate(TPA) on the formation of skin tumors in Charles River CD-1mice was studied. Mice were initiated with a single applicationof 0.2 µmol of 7, 12-dimethylbenz[a]anthracene (DMBA)in 0.2 ml acetone. Beginning two weeks after initiation, micewere treated twice weekly with various doses (0.01 – 20nmol) of TPA in 0.2ml acetone. Application of either 0.01 or0.1 nmol of TPA did not elcity tumors during the 50 weeks durationof treatment. A dose-dependent increase in the number of papillomaswas observed through the range of 1 to 10 nmol of TPA. Twiceweekly applications of 20nmol of TPA did not further enhancethe papilloma incidence. A good correlation was observed betweenthe induction of ornithine decarboxylase (ODC) activity andthe formation of skin tumors by various doses of TPA. To determine the effect of promotion duration on the incidenceof papillomas and carcinomas, mice were treated with 10 nmolof TPA for various durations (6,12,18,24,30, or 36 weeks) beginning2 weeks after initiation with 0.2 µmol of DMBA. Mice promotedfor only 6 weeks developed papilllomas and carcinomas afterpromotion had been discontinued. There was an intermediate incidenceof tumors in the group treated for 12 weeks. Promotion for 18,24, 30, or 36 weeks elicited virtually identical yields of papillomas.The incidence of carcinomas was proportional to promotion durationtimes of 6, 12, and 18 weeks, but carcinoma incidence was lessthan maximal in mice promoted for 24 weeks or longer. The results indicate that a) the incidence of papillomas servesas a rapid (18 weeks) index for subsequent appearance of carcinomas,b) twice weekly applications of 10 nmol of TPA for 18 weeksfollowing initiation of female CD-1 mice with 0.2 µmnolof DMBA is an appropriate protocol for maximum tumor yield ininitiation-promotion experiments, and c) ODC induction may bean important component of the mechanism of skin tumor promotionby TPA.     

The occurrence of tumours in F1, F2, and F3 descendants of pregnant mice injected with 7,12-dimethylbenz(a)anthracene

Pregnant MA mice were injected intraperitoneally with 400 micrograms of 7,12-Dimethylbenz(a)anthracene in olive oil during the last stages of pregnancy. The genetic design of the experiment included the follow-up of the descendants for three consecutive generations and the cross-mating between the F₁ descendants of treated mothers with descendants of untreated controls. An increased incidence of tumours was observed in the female F₁ and F₂ descendants and the female F_2f descendants (cross-mating of F₁ female descendants from a treated mother and male descendants from an untreated control). The tumours were observed in animals dying at a late age which approximately corresponded to the age when spontaneous tumours were observed in control animals. The most frequent types of tumours were the same as observed in the control animals, together with a handful of rarely observed tumours. In the F₁ and F₂ male descendants the tumour incidence was higher than in control animals but the difference was statistically not significant (binomial test). No significant increase in tumour incidence was observed in the F₃ and F_2m descendants (cross-mating of F₁ male descendants from treated mothers with female descendants from untreated controls). While the mice of F₁ generation were directly exposed to small amounts of the chemical carcinogen via the mothers' milk and excreta, and possibly through the placenta, it seems unlikely that DMBA as such was transmitted to the F₂ descendants.     

A study on skin tumour formation in mice with 50 Hz magnetic field exposure

In order to test the possibility that magnetic fields (MF) actas a tumour promoter, a long-term skin carcinogenicity studyof 50 Hz sinusoidal MF with flux densities of 50 µT and0.5 mT was performed in female NMRI mice. 7, 12–dimethylbenz[a]anthracene(DMBA) in acetone was applied to the dorsal skin, as an initiator,and exposure to MF was performed for 19 (weekdays) or 21 h/day(weekends and holidays) for 103 weeks starting one week afterthe initiator treatment. The phorbol ester 12–O–tetradecanoylphorbol-13-acetate(TPA) was used as a positive control for skin tumour promotingactivity. MF was also evaluated for complete carcinogenic actionin groups of mice that were treated with acetone only. Six animalsfrom each group were taken for skin hyperplasia analysis andwere killed after 9, 26 and 52 weeks. The appearance of skinlesions were carefully followed and histopathological diagnosiswas made for all neoplasms present at death. The statisticalanalyses on occurrence of skin tumour bearing animals and cumulatedskin tumours, with corrections for survival, did not reveala difference between the controls and the MF exposed groups.The epithelial thickness of DMBA + MF-treated animals was ofthe same magnitude as for DMBA-treated animals. Leukaemia wasa little more frequent among animals exposed to 0.5 mT MF comparedto the control animals. However this difference was not statisticallysignificant.     

Inhibition of both skin and lung tumorigenesis by Car-R mouse-derived cancer modifier loci.

The Car-R outbred mouse line was phenotypically selected for high resistance to two-stage skin tumorigenesis. In the present study we tested the hypothesis that a subset of genetic loci responsible for resistance to skin tumorigenesis of Car-R mice might also inhibit lung tumorigenesis. Skin and lung tumorigenesis were induced in groups of Car-R, SWR/J, (SWR/JxCar-R)F1 and SWR/Jx(SWR/JxCar-R) backcross mice by i.p. urethane initiation and skin TPA promotion. Car-R mice showed a much lower susceptibility to both skin and lung tumorigenesis as compared to SWR/J mice, which are susceptible to both lung and skin tumorigenesis. The Car-R-inherited genome significantly inhibited both skin and lung cancer development in the F1 progeny of Car-R with SWR/J mice. In the backcross population, skin and lung tumor phenotypes showed a statistically significant correlation, indicating that a subset of the cancer resistance alleles, which segregated in the Car-R line during selection for resistance to skin carcinogenesis, provides resistance to both skin and lung tumorigenesis.     

Increased multiplicity of lung adenomas in five generations of mice treated with benz(a)pyrene when pregnant

The effect of benz(a)pyrene (BP) given to female mice of A strain on the 18 and 19th days of pregnancy was studied in 5 consecutive generations. As expected there was a high incidence of lung tumours (TBA%) in the F1-generation of mice treated with BP (53.9 vs. 15.4% in control in females and 77.6 vs. 8.0% in control in males). The percentage of TBA was not increased (with one exception) in both males and females of F2-F5 generations which were not directly exposed to carcinogen. Tumor multiplicity increase occurred not only in the F1 generation of BP-treated mice but in both males and females of F2-F5 generations of mice which were not in direct contact with the carcinogen. This increase was statistically significant. There was a slight negative trend within F2-F5 generation of BP-treated mice which however was not significant. Thus the transgenerational carcinogenic effect manifested in a greater tumour multiplicity persisted for four unexposed generations.     

Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice

A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations.     

Study on prevention of two-stage skin carcinogenesis by Hibiscus rosa sinensis extract and the role of its chemical constituent, gentisic acid, in the inhibition of tumour promotion response and oxidative stress in mice.

We designed the present study to investigate the role of gentisic acid in the chemopreventive activity of Hibiscus rosa sinensis extract on 7,12-dimethyl benz(a)anthracene (DMBA)/croton oil-mediated carcinogenesis in mouse skin via 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced tumour promotion response and oxidative stress. Single topical application of DMBA followed by twice weekly applications of croton oil after one week for 20 weeks resulted in 100% incidence of tumours in animals in 15 weeks. However, application of H. rosa sinensis extract 30 minutes prior to the application of croton oil twice weekly for 20 weeks caused significant reduction in the number of tumours per mouse and the percentage of tumour-bearing mice. Also, the latency period for the appearance of the first tumour was delayed on H. rosa sinensis pretreatment. A single topical application of TPA caused significant depletion in reduced glutathione (GSH) content, activities of its metabolizing and antioxidant enzymes, while malondialdehyde (MDA) formation, H2O2 content, ornithine decarboxylase (ODC) activity and DNA synthesis were significantly increased. Interestingly, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/kg body weight) and gentisic acid (2.0 microg and 4.0 microg/0.2 ml acetone per animal) restored the levels of GSH, and its metabolizing and antioxidant enzymes (P<0.05). There was also a statistically significant reduction in MDA formation and H2O2 content (P<0.05) at both doses. Although inhibition of ODC activity by gentisic acid was not dose-dependent, thymidine incorporation in DNA (P<0.05) was dose-dependently recovered by the plant extract and its chemical constituent. We therefore propose that gentisic acid has a role in the modulatory activity of H. rosa sinensis extract.     

Effect of colchicine injection prior to the initiating phase of two-stage skin carcinogenesis in mice.

Colchicine injected 5, 9 and 24 h respectively before initiation (using s.c. injection of urethane for initiating action and TPA skin applications for promoting action, in female ICR mice) led to a significant increase in skin tumour incidence in the --9-h group, and an increase in percentage malignancy in both the --5- and --9-h groups. These times corresponded to the peak of metaphase arrest by the colchicine. The results are discussed in relation in those of Pound and Withers (1963) and others, who found that mitotic stimulation at the time of urethane initiating action raised the ultimate tumour incidence; and the inference is drawn that initiating action in mouse skin may occur during the M phase, rather than during the G1, S, or G2 phases, as suggested by others.