Application Of Croton Oil Research Articles

Two-stage skin carcinogenesis in sensitive and resistant mouse strains.

Strains of laboratory mice vary markedly in their susceptibility to two-stage skin carcinogenesis using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating carcinogen and croton oil as promoter. This study has been undertaken in order to clarify the basis of the strain differences. LACA mice were used as the susceptible strain and BALB/c mice as the resistant strain. DMBA was a more effective complete carcinogen in LACA mice than in BALB/c mice. However, dose-response studies with respect to DMBA in the two strains in two-stage carcinogenesis suggested that metabolic activation of DMBA to the active carcinogen was not limiting in the resistant strain. The observed strain differences in response to DMBA in one and two stage carcinogenesis may reflect the ability of DMBA to also act as a promoter in the two strains. The possibility that the strains vary in their ability to repair damaged DNA has, however, not been eliminated. Unlike polycyclic aromatic hydrocarbon carcinogens such as DMBA, phorbol ester promoters (the active components in croton oil) do not appear to require metabolic activation. However, they are degraded and inactivated by epidermal and other cells. Experiments in which the dose and frequency of application of croton oil were increased, and the unrelated promoter anthralin was substituted for croton oil, failed to produce any evidence that differences in promoter degradation contributed to the differences in susceptibility between strains.

The Effect of Various Sunscreen Agents on Skin Damage and the Induction of Tumor Susceptibility in Mice Subjected to Ultraviolet Irradiation

Sunscreen preparations containing various chemical UV absorbers, para-aminobenzoic acid (PABA), 2 PABA derivatives, benzophenone or a combination of these were topically applied to the backs of C3H/HeN mice prior to their being irradiated with ultraviolet light in the UVB range. In all cases this treatment was effective in preventing the pathological skin changes associated with UVB irradiation. Histological evaluation of skin biopsies from mice treated with the sunscreen preparations and UVB irradiation showed little or no difference from normals in amount of hyperplasia, melanization or parakeratosis present. These histologic changes were observed in animals receiving UVB irradation in the absence of any sunscreen agent. Pretreatment with the various sunscreen agents did not, however, prevent the induction of tumor susceptibility as measured by the sustained growth of a UV-induced tumor which is immunologically rejected in normal syngeneic mice. These data show a clear distinction between the effects of UVB irradiation leading to histological changes in the epidermis and those leading to the state of tumor susceptibility in mice. The distinction was further corroborated by the finding that epidermal hyperplasia induced by repeated applications of croton oil had no significant enhancing or inducing effects on the induction of tumor susceptibility. In addition, the induction of tumor susceptibility. In addition, the induction of tumor susceptiblity is not due to wavelengths of light less than 320 nm since this effect was abrogated when the UVB radiation was filtered through glass. Possible mechanistic differences between the tumor susceptiblity generated in UVB and photoprotected UVB irradiated animals were observed, however, when we attempted to adoptively transfer the state of tumor susceptibility to normal animals. While it was readily transferable with splenic lymphoid cells from UVB irradiated animals, all attempts to transfer the tumor susceptibility from photoprotected animals have, to date, been unsuccessful.

Measurement and differentiation of the cellular infiltrate in experimental toxic contact dermatitis

Toxic (irritant) contact dermatitis was elicited by epicutaneous application of dinitrochlorobenzene (DNCB) and croton oil in unsensitized guinea pigs. The course of the skin reaction was studied with the naked eye, low-power microscopy, and a method based on counting of infiltrating cells in the upper corium. A weakly toxic dose of DNCB (100 microgram/cm2) gave marked erythema and moderate swelling, more pronounced 6 h after application of the DNCB than at 24 h or 48 h. A significant increase in mononuclear cells in particular but also in neutrophil and eosinophil granulocytes was noted in the upper corium. A more strongly toxic dose (500 microgram/cm2) gave a similar visible reaction, this too most marked at 6 h, but here there was an increase in neutrophil granulocytes in particular. Over and above predominant mononuclear-cell infiltration, croton oil (10 microgram/cm2) caused a slight increase in basophil cells. The reaction thus resembled a contact allergic reaction. In the light of earlier findings in allergic contact reactions the results suggest that weak stimuli, toxic or allergenic, elicit a non-specific skin response. With stronger stimulation the histological picture is modified and the cellular response acquires a pattern characteristic of toxic or allergic contact dermatitis.

Change of polyamine content in mouse skin by leupeptin, a protease inhibitor, during early stage of tumorigenesis.

The effect of a microbial protease inhibitor, leupeptin, on the content of polyamine in the mouse skin was examined during the early stage of tumorigenesis induced by a single application of 7,12-dimethylbenz[a]anthracene (DMBA) and repeated application of croton oil thereafter. Polyamine content in the skin was measured at 3, 5, 7, and 9 weeks during tumorigenesis. The mice with no visible tumor were selected for measurement of polyamine content at 9 weeks. Mice were left untreated for at least 1 week before measurement of polyamine. Polyamine in the skin was extracted with ice-cold 0.4N HClO4 and separated into putrescine, spermidine, and spermine fractions through CM-cellulose column. Polyamine concentration was determined by fluorometry with fluorescamine. Group A mice painted with croton oil 3 times a week did not develop tumors. Group B mice painted with a single DMBA developed skin tumors, and group C mice painted with a single DMBA and croton oil 3 times a week showed higher development of skin tumors than group B. Group D mice treated as group C and then painted with leupeptin about 2 hr after croton oil treatment. Animals in groups B, C, and D had higher spermidine content as group A at 3 and 5 weeks. Content of spermidine in group B decreased at 7 and 9 weeks compared with group C which had a high content throughout the time tested. Leupeptin treatment in group D inhibited spermidine content in the skin after 7 weeks without affecting until 5 weeks.

Lack of effect of trichloropropene oxide on benzo(a)pyrene tumor-initiating activity on mouse skin

The potent epoxide hydratase (EH) inhibitor, 1,1,1,-trichloro- 2,3-propene oxide (TCPO), did not enhance the tumor-initiating ability of benzo(a)pyrene (BP) when applied simultaneously with BP in a two-stage system of tumorigenesis in male Swiss mice. BP ( 0.1 μmole) with or without TCPO ( 2 μmole) was applied either once (day 1 or twice 1 and 3 to the skin of the backs of mice shaved 24 hr before; thereafter the mice were given weekly applications of croton oil ( 50 μl1% in acetone per mouse for 50 weeks). Skin tumors started to appear 15 weeks after initiation of the mice, and at the same time in all groups. The experiment was terminated 50 weeks after application of carcinogen. The skin microsomal epoxide hydratase ( 48–52 pmole BP- 4,5 diol/min/mg) was inhibited 3 hr after application of BP alone or BP plus TCPO. No inhibition was observed in microsomes isolated 16 or 24 hr after application of carcinogen or carcinogen plus epoxide hydratase inhibitor. TCPO has no in vitro effect on the 3H -BP binding to epidermal DNA 24 hr after application of benzo(a)pyrene. Rapid trapping of TCPO by intracellular glutathione and/or nucleophiles would explain this absence of effect, which contradicts the results of D. L. Berry and other ( J. Nat. Cancer Inst. Vol. 58, No. 4, 1051–1055, 1977).

Early fine structural changes in human epidermis following application of croton oil

Early epidermal changes in human subjects following application of 10% croton oil in acetone were studied by electron microscopy. The prominent changes observed at 1 and 5 hours were degeneration of epidermal cells and intercellular edema in the basal and spinous layers. One type of degeneration was homogenization and aggregation of the tonofilaments of keratinocytes at 1 hour, which may explain the presence of dyskeratotic cells at 5 hours. Another type was of a cytolytic nature as evidenced by severe perinuclear edema of keratinocytes and destruction of Langerhans' cells with outpouring of Birbeck's granules in the intercellular spaces.

Effects of Cellophane Tape Stripping of Mouse Skin on Epidermal Growth Regulators (Chalones)

Variations in epidermal chalones after a single surface application of methylcholanthrene and croton oil have been described in previous papers. This paper reports a study of the effect of adhesive tape stripping of the skin on epidermal growth regulators (G1 and G2 chalones). Pieces of adhesive tape were applied 6 times to the same area of skin in groups of mice. The short-term effects of tape stripping on epidermal DNA synthesis and on mitotic rate were studied at different intervals after stripping. Other groups of mice were killed at similar time intervals after stripping, and the treated area of skin was homogenized and extracted with water. The inhibitory effect of these extracts on normal epidermal DNA synthesis and mitotic rate was assayed in normal hairless mice. The resulting inhibitions were interpreted as an expression of the concentration of G1 or G2 chalone in the skin extracts. The first experiment confirmed that cellophane tape stripping gives rise to a short block in epidermal mitotic activity and probably also in DNA synthesis. This was followed by bimodal peaks of increased activity, the two maxima of labelling index being found on days 2 and 6, and the two maxima of mitotic rate on days 1-2 and 7. The concentrations of the two chalones in the skins of treated animals varied in inverse proportion to the alterations in the DNA synthesis and the mitotic rate, with one exception. Here there was initially a depression of the mitotic rate and a low concentration of G2 chalone. This was interpreted as a short reaction of the basel cells to the stripping trauma. It is concluded that adhesive tape stripping removes the differentiating cells and injures some basel cells, simultaneously altering the content of G1 and G2 chalones. The resulting increase in the rates of DNA synthesis and mitosis lasts only until the number of cells is high enough to produce growth-regulating substances (chalones) again. This theory may explain the changes observed. Since similar reactions are seen after both carcinogenic and co-carcinogenic chemical injury of the epidermis, the reaction pattern seems to be a general response to cell injury or cell removal.

Localization Of Adenosine 3', 5'-Monophosphate In Mouse Epidermis By Immunofluorescence

Adenosine 3',5'-monophosphate (cyclic AMP) has been localized in mouse epidermal cells using an immunofluorescent technique. Within 10 min following the intraperitoneal injection of isoproterenol or 30 hr following the topical application of croton oil in acetone, staining was clearly visible in the cytoplasm of the basal cells.

Transformation of C3H/10T1/2CL8 mouse embryo fibroblasts by ultraviolet irradiation and a phorbol ester.

ULTRAVIOLET light is the major cause of human skin cancer, and papillomas and malignant carcinomas have been induced in the skins of rats and mice by repeated exposure to it1–5. Pound6 reported that although a single short exposure of mouse skin to ultraviolet light did not induce any tumours, when the single exposure was followed by repeated applications of croton oil, many tumours were induced. Thus ultraviolet light acted as an initiator in the two-stage process of skin tumour development7,8. X rays can induce oncogenic transformation of cells in culture9,10, and a preliminary report has appeared on the transformation of hamster embryo cells by ultraviolet light11.

Effects of Croton Oil on Epidermal Growth Regulators (Chalones)

Variations in epidermal chalones after a single surface application of methylcholanthrene have been described in previous papers. This paper reports a study of the effect of croton oil on epidermal growth regulators (G1 and G2 chalones). Hairless mice received a single topical application of 0.2 ml 0.25% acetone solution of croton oil. Control mice received only acetone. The short-term effect of croton oil on epidermal DNA synthesis and mitotic rate was studied. Other groups of croton oil-treated and acetone-treated mice were then killed at similar time intervals, and the treated area of skin was homogenized and extracted with water. The inhibitory effect of these extracts on normal epidermal DNA synthesis and mitotic rate was assayed in normal hairless mice. The resulting inhibition was interpreted as an expression of the concentration of G1 and G2 chalones, respectively, in the skin extracts. The first experiment confirmed that a single croton oil application provokes a short block in epidermal mitotic activity and probably also in DNA synthesis. This was followed by bimodal peaks of increased activity, the two maxima of mitotic rate on days 2 and 7. The concentration of the two chalones in the skins of treated animals varied in inverse proportion to the alterations in the DNA synthesis and the mitotic rate, with one exception. There was here initially a depression both of the mitotic rate and a low concentration of G2 chalone. This was interpreted as a short, initial direct effect of croton oil on the G2 chalone present at the time of application. It is concluded that croton oil application injures and kills epidermal cells, with subsequent alterations in the content of G1 and G2 chalones. This theory may explain the changes observed. The effects of croton oil on the amount of G1 and G2 chalones in the skin are probably related to the direct, toxic, cell-killing effect of croton oil, and not to its specific cancer promoting potency.

Studies on the Modifying Effect of Ultraviolet Radiation on Chemical Skin Carcinogenesis

The effect of ultraviolet light on chemically-induced skin carcinogenesis in mice was studied. Ultraviolet (UV) irradiation (5.5 × 10 7 ergs/cm 2 ) 24 hr before repeated applications of 7,12-dimethylbenz[a]anthracene (DMBA) resulted in an increased tumor yield, while irradiation 1 hr after painting caused a decrease. UV irradiation prior to a single application of 3,4-benzo[a]pyrene as an initiating agent, followed by repeated applications of croton oil, increased the neoplastic response. The enhancing effect of a low, nonulcerating dose of UV light 2.8 × 10 6 ergs/cm 2 on initiation by DMBA in two-stage skin carcinogenesis was also significant.

Ultraviolet light irradiation as initiating agent in skin tumor formation by the two-stage method

The biological and morphological characteristics of neoplastic progression in mouse skin induced by the two-stage method, using ultraviolet light as an initiator and croton oil as a promoter, were studied by biological and histological methods. Ultraviolet light proved to be an effective initiator in skin tumor formation, when followed by repeated applications of croton oil. In comparison with experiments using 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator, the average latent period was longer and the number of tumor-bearing animals and of tumors was lower. Morphologically, an orderly epidermal hyperplasia, progressing through papillomatous hyperplasia and ending in extroverted squamous cell papillomas, occurred in only a few animals. The proliferation of epithelial cells induced by multiple u.v. irradiation was disorderly with distinct cytological and histological abnormalities. A few squamous cell carcinomas developed from the borders of ulcers induced by repeated u.v. light applications. Efforts to increase the neoplastic response, by giving either u.v. light 10 times prior to promotion or croton oil immediately after each of 16 u.v. irradiations, did not increase the total number of tumors but did induce some malignant ones. These studies emphasize the specificity of initiation and promotion in skin tumor formation and the different actions of physical and chemical carcinogens on animal skin, depending upon the combination of progression and persistence of destructive and proliferative actions specific to each agent.

Fast onset of DNA synthesis stimulated by tumor promotor in mouse epidermis at the initation stage of carcinogenesis.

Mouse skin was treated with 7,12- dimethylbenz[a]anthracene (DMBA), benzo[a] pyrene (BP), or urethan. After 5 weeks, on the same region of skin, 0.5, 0.1, or 0.02% croton oil was applied. 3H-thymidine (3H-TDR) was injected every 4th hour during a 20- to 24-hour period after application of croton oil. The labeling index in interfollicular epidermis was determined autoradiographically. Croton oil induced transition to the DNA synthesis phase in most basal cells. The mean time between application of croton oil and transition of basal cells to the DNA synthesis phase was significantly shorter in DMBA-“initiated” epidermis than in normal epidermis for 3 concentrations of croton oil. The labeling index for mature cells also increased more rapidly, in epidermis treated with initiator. The difference between normal and initiated epidermis was most pronounced after 0.1% croton oil was applied. With this concentration, initiation with BP and urethan also shortened the time between stimulation and onset of DNA synthesis. Without stimulation, the labeling index curves for basal cells did not differ in normal and initiated epidermis on repeated injections of 3H-TDR. Fast onset of stimulated DNA synthesis possibly is characteristic of epidermis treated with initiators and results from increased sensitivity of the genome to tumor promoters.

Quantitative evaluation of antiphlogistic effect of corticosteroids topically applied

Summary The adrenal ascorbic acid depletion, elicited in rats by conjunctival phlogosis provoked by application of croton oil, has been assumed as a test for quantitative evaluation of antiphlogistic effect of the following topically applied steroids: hydrocortisone acetate, triamcinolone acetonide, dexamethasone valerate. It is shown that the ascorbic acid depletion is reduced by such steroids and that such a reduction is linearly related to the log of their increasing concentrations. The parallelism of the calculated regression lines enables the evaluation of their antiphlogistic potency in reference to hydrocortisone acetate, which is 3.80 for triamcinolone and 4.27 for dexamethasone. The results obtained are compared with those obtained by the other experimental methods devised in order to evaluate the topical antiphlogistic effect of corticosteroids.

Prophylaxis of chemically induced benign and malignant epithelial tumors by vitamin A acid (retinoic acid)

The prophylactic effect of retinoic acid on the development of chemically induced papillomas and carcinomas of the skin of mice was examined. The tumors were induced by local application of dimethylbenzanthracene and croton oil. Retinoic acid— 200 mg/kg every 14 days—was administered orally during the promotion phase of carcinogenesis. It delayed the appearance, retarded the growth and lead to regression of papillomas. The induction of carcinomas was also inhibited. By prophylactic administration of retinoic acid the appearance of carcinomas was delayed and their incidence reduced.

The stimulation of phospholipid metabolism in mouse skin following phorbol ester treatment

The incorporation of radioactive precursors into mouse skin phospholipids was examined after a single application of either croton oil or its most active component, 12- O-tetradecanoylphorbol 13-acetate. 1. 1. The incorporation of 32P 1 into all phospholipids is stimulated by either croton oil or 12- O-tetradecanoylphorbol 13-acetate but not by the parent compound phorbol. The stimulation of incorporation into phosphatidylcholine is largest and exhibits a biphasic pattern with two distinct peaks of incorporation. The first peak occurs 4–6 h after treatment while the second peak is observed at 2 days. 2. 2. Incorporation studies using [ ME- 14C]methionine and [ ME- 14C]choline suggest that the first peak represents synthesis of phosphatidylcholine through the methylation of phosphatidylethanolamine and the cytidinediphosphocholine pathway, while the latter peak involves synthesis mainly through the cytidinediphosphocholine pathway. 3. 3. During the first peak of the response an accumulation of phosphatidylcholine occurs due to either an increased permeability of cell membranes to plasma phosphatidylcholine or the activation of mouse skin enzymes which synthesize phosphatidylcholine. 4. 4. Pretreatment of mouse skin with cycloheximide before 12- O-tetradecanoylphorbol 13-acetate application inhibits labeled leucine incorporation into protein but has little effect on the early stimulation of 32P 1 incorporation into phosphatidylcholine. 5. 5. These early alterations in phospholipid metabolism after phorbol ester treatment support the concept that the effects of these phorbol esters are mediated through membrane interactions.

Hydroxyurea: Possible Relationship of Cytotoxicity to Inhibition of Skin Carcinogenesis<xref ref-type="fn" rid="FN2">2</xref>

Intraperitoneal injection of mice with hydroxyurea (0.5 mg/g body wt) resulted in the loss of about 60% of the DNA-synthesizing cells from normal mouse epidermis. The same dose of hydroxyurea was not cytotoxic in rapidly proliferating epidermis 1 day after croton oil application. When a second hydroxyurea injection was given 2 days after croton oil, about 30% of the DNA-synthesizing cells were destroyed. Inhibition of skin carcinogenesis by hydroxyurea may be due to its toxicity to potential tumor cells.

Polyinosinic-polycytidylic acid inhibits chemically induced tumorigenesis in mouse skin.

Polyinosinic-polycytidylic acid administered intraperitoneally inhibits (i) the formation of skin tumors induced by a single topical application of 9,10-dimethylbenzanthracene followed by weekly applications of croton oil and (ii) the formation of skin tumors induced by a single large application of 9,10-dimethylbenzanthracene.

Induced cell proliferation and the initiation of skin tumour formation in mice by ultraviolet light

A single short exposure of the skin of mice to ultraviolet light did not produce any tumours, but a significant number of tumours developed when the exposure was followed by repeated applications of croton oil. Ultraviolet radiation therefore behaves as an initiating agent, resembling this action of some other carcinogens for the skin of mice. Treatment of the skin of the mice with acetic acid, xylene or croton oil 20 hours before irradiation increased the number of tumours. It is considered that cells induced to proliferate are more susceptible to the carcinogenic effect of ultraviolet radiation than their normal counterparts. A single short exposure of the skin of mice to ultraviolet light did not produce any tumours, but a significant number of tumours developed when the exposure was followed by repeated applications of croton oil. Ultraviolet radiation therefore behaves as an initiating agent, resembling this action of some other carcinogens for the skin of mice. Treatment of the skin of the mice with acetic acid, xylene or croton oil 20 hours before irradiation increased the number of tumours. It is considered that cells induced to proliferate are more susceptible to the carcinogenic effect of ultraviolet radiation than their normal counterparts.

The influence of preliminary irritation by acetic acid or croton oil on skin tumour production in mice after a single application of dimethyl-benzathracene, benzopyrene, or dibenzanthracene.

Groups of mice were given a single application of acetic acid to one side of the skin of the back. Other groups were given an application of croton oil to the whole area of the skin of the back, a control group had no application of croton oil. At the same time as, 24 hours, 72 hours, or 9 days after the application of acetic acid or croton oil, the mice were given a single application of one of the carcinogenic hydrocarbons DMBA, BP or DBA, to the whole area of the skin of the back. The number of tumours produced was greater in areas that had the preliminary treatment with acetic acid or croton oil at the same time or 24 hours before the carcinogen. There was a doubtful effect at an interval of 3 days and no effect at 9 days. It is considered that proliferating epidermal cells are more susceptible to the action of the carcinogens perhaps during replication of DNA.