A Comparison of Three Cytotoxicity Endpoints in the Corneal HCE-T Model.

The prediction of ocular irritation potential from in vitro assays still presents a problem, despite a number of validation trials. A study with coded cosmetic formulations, for which historic in vivo data were available, has been conducted with a human corneal multi-layered model system. This corneal model, the HCE-T model, was developed by using HCE-T cells, a transfected human corneal epithelial cell line. The relative effectiveness of three endpoints that provide a measure of cytotoxicity in the HCE-T model was evaluated. Cell viability immediately after exposure to the test materials was determined by using the MTT and Alamar Blue™ (AB) assays, and, 24 hours later, by using the MTT, AB and lactate assays. Viability measurements with the MTT, AB and lactate assays gave similar dose-response curves at the 24-hour endpoint. One formulation (an anti-dandruff shampoo) caused a less severe drop in viability in assays conducted immediately after the exposure than at the 24-hour time-point. There was little deterioration in viability with the other test materials. The ranking of the test formulations on the basis of relative loss of viability and release of lactate resulted in the same order as for the Modified Maximum Average Draize Test Score. Comparison of the HCE-T model cytotoxicity assay results with historic in vitro data from two different cytotoxicity assays, conducted by using fibroblast monolayer cultures and the same materials, indicated that the multi-layered corneal model had a greater predictive ability. The results of a blind trial with the lactate assay in two laboratories indicated that the techniques required were transferable between laboratories. The lactate results were reproducible between laboratories, even when cultures derived from different passage human corneal cells were tested, provided that the passage number was below 20.

Evaluation of a human corneal epithelial cell line as an in vitro model for assessing ocular irritation.

A human corneal epithelial cell line, 10.014 pRSV-T (HCR-T cells), has been used to develop a three-dimensional in vitro model of the human corneal epithelium (HCE-T model). HCE-T cells form a stratified culture when grown at the air-liquid interface on a collagen membrane in serum-free medium. This model served as the basis for assays which supported the ocular irritancy assessment of water-soluble test substances. Cellular alterations in the HCE-T model were measured following 5-min topical exposures to 20 chemicals [listed in the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) Reference Chemicals Data Bank] and 25 surfactant-based product formulations [utilized in the Cosmetic, Toiletry, and Fragrance Association (CTFA) Alternatives Program Phase III]. In vitro assays used were transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). These measured alterations in the barrier function of this corneal epithelial equivalent. Barrier function is a well-developed property in the HCE-T model that supports the mechanistic relevance of these assays. In vitro data, averaged from replicate assays, were compared to respective Draize rabbit eye irritation data from the publicly available ECETOC and CTFA databases using linear regression with Pearson's correlation analysis. For chemicals, Pearson's correlation coefficients, r, from comparisons of Draize maximum average scores (MAS) to TEP and TER data were 0.71 and 0.55, respectively. For product formulations, Pearson's correlation coefficients from comparisons of Draize MAS to TEP and TER data were 0.86 and 0.80, respectively. Data indicated that barrier function alterations in the HCE-T model correlated with ocular irritancy and corneal toxicity. While the irritancy of the chemicals tested was effectively assessed only by the TEP assay, that for the surfactant-based product formulations was effectively assessed by both the TEP and TER assays. Results also suggested that the HCE-T TEP and TER assays vary in their effectiveness for evaluating specific classes of test materials.

IRAG working group 5. Other assays. Interagency Regulatory Alternatives Group.

As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5. "Other Assays") were the EYTEX assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.

Inhibition of chrysarobin skin tumor promotion in SENCAR mice by antioxidants.

The present study was designed to further investigate the role of reactive oxygen species in the mechanism of action of anthrone tumor promoters. To accomplish this, the effects of several antioxidants on the induction of epidermal ornithine decarboxylase (ODC) activity, epidermal hyperplasia, skin edema, and skin tumor promotion by chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) were tested. Ascorbyl palmitate (AP), given 5 min prior to the promoter at 1 and 4 mumol doses, effectively inhibited the induction of ODC activity (28% and 59%, respectively) by 220 nmol of chrysarobin. Using a similar protocol, alpha-tocopherol acetate (alpha-TA) at 10 and 40 mumol doses also effectively inhibited the induction of ODC activity (36% and 70%, respectively) by 220 nmol of chrysarobin. In contrast, butylated hydroxyanisole (BHA) at doses up to 56 mumol per mouse was ineffective at inhibiting the induction of ODC by chrysarobin. AP at the 4 mumol dose significantly inhibited the induction of edema by chrysarobin by 24% and the induction of epidermal hyperplasia by 23%. alpha-TA at the 40 mumol dose also significantly inhibited chrysarobin-induced edema by 22% and epidermal hyperplasia by 17%. Skin tumor promotion in mice initiated with 25 nmol of 7,12-dimethylbenz[a]anthracene and promoted with once-weekly treatments of 220 nmol chrysarobin was markedly inhibited by treating mice with either AP or alpha-TA 5 min prior to promoter treatment. AP at 1 and 4 mumol doses significantly reduced the number of papillomas per mouse, by 48% and 44%, respectively. alpha-TA at 10 and 40 mumol doses also significantly reduced the number of papillomas per mouse, by 33% and 59%, respectively. In two separate tumor experiments, BHA at 2.8 and 5.6 mumol failed to inhibit chrysarobin tumor promotion. The current results provide further support for a role of reactive oxygen species in the tumor promoting activity of anthrones. In addition, the data indicate that the phenolic antioxidant BHA is an ineffective inhibitor of anthrone tumor promotion.

Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium.

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca2+ concentrations (Cao) from 0.05 mM to > or = 0.1 mM. The addition of Sr2+ (> or = 2.5 mM) to medium containing 0.05 mM Ca2+ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Cao to 0.12 mM. Sr2+ in 0.05 mM Ca2+ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr2+ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca2+ in the medium, the Sr2+ effects are reduced, and Sr2+ is ineffective when both Ca2+ and serum are deleted from the medium. Sr2+ treatment increases the ratio of fluorescence intensity of the intracellular Ca2+ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca2+ and/or Sr2+. At doses sufficient to induce differentiation, Sr2+ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of 85Sr2+ by primary keratinocytes are similar to those of 45Ca2+. At low concentrations, the initial uptake of both 45Ca2+ and 85Sr2+ reaches a plateau within 1 hr; at higher concentrations, the uptake of both 45Ca2+ and 85Sr2+ increases continuously for 12 hr. In keratinocytes pre-equilibrated with 45Ca2+ in 0.05 mM Ca2+ medium, Sr2+ causes an increase of 45Ca2+ uptake, which is dependent on the presence of serum. These results suggest that Sr2+ utilizes the same signalling pathway as Ca2+ to induce keratinocyte terminal differentiation and that Ca2+ may be required to exert these effects.

Regulation of intracellular free calcium in normal murine keratinocytes.

Cultured normal murine keratinocytes maintain a basal cell phenotype in medium with a Ca2+ concentration of 0.05 mM and differentiate when exposed for 28-48 h to medium supplemented with extracellular Ca2+ greater than 0.10 mM. Previous studies have documented Ca2+ activation of signaling pathways in the plasma membrane and tightly regulated cellular responses to small incremental changes in extracellular Ca2+. To determine if changes in free cytosolic calcium (Cai) are associated with these early signaling events, digital image analysis of fura-2-loaded keratinocytes was used to measure Cai in individual cells. Basal keratinocytes in 0.05 mM Ca2+ display a biphasic Cai increase when exposed to greater than 0.1 mM Ca2+ in serum-containing medium. These separate phases were controlled by different media components. Initial peak Cai occurred rapidly (within 60 s), was transient (lasting less than 5 min), and resulted from release of 10-20% of total intracellular Ca2+ stores. Peak Cai depended on serum concentration and was independent of extracellular Ca2+. This transient Cai response was lost as keratinocytes differentiated. Plateau Cai level was sustained (greater than 24 h) and depended on extracellular Ca2+, but not serum. The magnitude of plateau Cai increased incrementally following increases in extracellular Ca2+ as small as 0.02 mM. A similar biphasic Cai increase was noted in cultures of murine dermal fibroblasts stimulated by 1.2 mM Ca2+ and serum. However, fibroblasts did not lose the serum response in high-Ca2+ medium, and plateau Cai was not sensitive to small changes in extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the regulation of intracellular calcium in normal and neoplastic keratinocytes are not caused by ras gene mutations.

The development of resistance to terminal differentiation is an early event in epidermal neoplasia. Altered differentiation can be detected in vitro since normal epidermal cells are induced to differentiate in medium with Ca2+ greater than 0.1 mM while neoplastic epidermal cells and keratinocytes transduced with a v-rasHa gene are resistant to Ca2+. In normal epidermal cells, the elevation of extracellular Ca2+ (Cao) from 0.05 to 1.2 mM causes a biphasic intracellular Ca2+ (Cai) response in which a transient (10 min) peak of 4-5-fold over basal values is followed by a sustained (greater than 24 h) 2-fold increase in steady-state Cai. The transient peak in Cai is dependent on a serum component and independent of Cao, while the sustained plateau is directly dependent on Cao. The transient peak responding to a serum factor is lost in normal cells after 24 h in 1.2 mM Ca2+, a time when these cells are differentiating. Two neoplastic keratinocyte cell lines, SP-1 and 308, which produce benign tumors in vivo, also have a biphasic Cai response to an increase in Cao. In these cells, the transient peak is also serum dependent and amplified to 10-fold over basal values. However, the plateau value is not sustained and returns to basal values by 8 h, independent of Cao. Furthermore, 308 cells remain sensitive to the serum-induced Cai transient after 24 h in 1.2 mM Ca2+. To determine whether the activating c-rasHa mutation in 308 and SP-1 cells was responsible for the altered Cai regulation, a v-rasHa gene was introduced into normal keratinocytes by a defective retrovirus. This also produces the papilloma phenotype in vivo. Recipient cells were resistant to Ca(2+)-induced terminal differentiation although they did not proliferate in 1.2 mM Ca2+. The Cai profile in response to 1.2 mM Ca2+ was identical in normal and v-rasHa keratinocytes, and these cells lost the serum-induced transient Cai peak after 24 h. Thus, the activation of the c-rasHa gene in 308 or SP-1 cells is probably not solely responsible for the altered Cai response in neoplastic cell lines. Sustained physiological elevation of Cai may be relevant to the loss of proliferative potential in both normal and v-rasHa keratinocytes in 1.2 mM Ca2+. In addition, v-rasHa-mediated or activated c-rasHa-mediated changes in a complementary pathway may contribute to the block in terminal differentiation in neoplastic cells.

Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes.

Normal keratinocytes proliferate when cultured in medium with 0.02-0.10 mM calcium and terminally differentiate when medium calcium is increased to greater than 0.1 mM. In contrast, neoplastic keratinocyte cell lines maintain the potential for continued cell renewal and survive when external calcium is increased. In order to determine whether elevation of extracellular calcium produced changes in intracellular free calcium (Cai) levels, Cai was measured in individual living keratinocytes by use of the fluorescent calcium probe fura-2. Most normal keratinocytes responded to increased extracellular calcium by a gradual 2- to 3-fold increase in Cai lasting for at least 28 min. A subpopulation displayed a sharp peak of Cai at 2 min. In contrast, the Cai level in neoplastic cells in either low or high calcium medium was 2- to 3-fold higher than that in normal cells, and all cells in the population showed a transient 4- to 9-fold elevation of Cai 2 min after external calcium was increased. Thus normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium. The regulation of Cai in keratinocytes may be important in determining their potential for terminal differentiation.

Histologic alterations produced by chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) in SENCAR mouse skin: relationship to skin tumor promoting activity.

Histologic changes induced in SENCAR skin following a single treatment with chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) exhibited differences in time course from that observed with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although not significantly different, maximum elevations in epidermal thickness, total number of nucleated epidermal cells, and dark basal keratinocytes (DCs) induced by 220 nmol chrysarobin occurred at 96 h after treatment, while those induced by 3.4 nmol TPA occurred at 48 h. Both compounds elicited comparable inflammatory responses. Twice-weekly applications of chrysarobin for 2.5 weeks induced a moderate hyperplasia, increase in total nucleated epidermal cells, and increased DCs at 48 and 96 h after the last treatment, with a higher value for these parameters occurring at 48 h. Interestingly, the magnitude of these changes was similar to that observed after a single application. In contrast, twice-weekly applications of TPA induced a dramatic, potentiated induction of epidermal hyperplasia and DCs. Once-weekly applications of chrysarobin led to a potentiated induction of both hyperplasia and DCs compared to the twice-weekly treatment regimen and also more effectively promoted epidermal papillomas in previously initiated SENCAR mice. Skin sections from mice treated with chrysarobin displayed overt signs of epidermal toxicity including altered basal cell morphology and a decreased number of basal cells per 125 micron of basement membrane. Hyperplasia induced by multiple but not single treatments with chrysarobin and TPA correlated quantitatively with their papilloma promoting activity. In addition, the data suggest that epidermal toxicity may play a role in tumor promotion by anthrones.

Tumor progression in Sencar mouse skin as a function of initiator dose and promoter dose, duration, and type.

The influence of initiator dose and promoter dose, duration, and type on the progression of papillomas to carcinomas was examined in Sencar mice. A good dose-response relationship for promotion of papilloma formation by 12-O-tetradecanoylphorbol-13-acetate (TPA) [following initiation with 6.5 micrograms of 7,12-dimethylbenz(a)anthracene (DMBA)] was observed in the range of 0.125 to 2.0 micrograms/mouse. A maximal papilloma response was induced with 2 micrograms/mouse (24 papillomas/mouse). When adjusted for mortality, the carcinoma incidence after 60 wk of promotion was essentially the same (approximately 80%) for doses above 0.5 micrograms/mouse. In a related experiment, mice were given an initiation dose of either 2 or 20 micrograms of DMBA followed by applications of 2 micrograms of TPA for 3, 5, 7, or 60 wk. Papilloma formation was proportional to length of treatment, with a maximum of 29 papillomas/mouse (20-micrograms initiating dose of DMBA) and 10 papillomas/mouse (2-micrograms initiating dose of DMBA) occurring between 10 and 15 wk of promotion. In this experiment, the carcinoma incidence was clearly proportional to the duration of promoter treatment at the low initiation dose of DMBA. The carcinoma incidence, on the other hand, was similar (approximately 70%) in groups of mice given an initiation dose of 20 micrograms of DMBA and promotion treatment for greater than or equal to 5 wk. Thus, the initiator dose had a dramatic effect on the outcome of these experiments. Additional experiments were performed to compare tumor progression with the anthrone promoter, chrysarobin. At optimal promoting doses, chrysarobin treatment produced a maximum number of papillomas that was approximately 1/3 that produced by TPA (6.4 versus 17.0 papillomas per mouse, respectively). However, the carcinoma response was very similar in these two treatment groups, confirming previous work from this laboratory. In addition, chrysarobin treatment following 10 wk of TPA promotion did not enhance the progression of preexisting papillomas to carcinomas. The data presented in this paper are consistent with a model in which several types or stages of papillomas are initially produced during two-stage carcinogenesis in mouse skin with different probabilities of progressing to carcinomas. However, the data indicate that optimal doses of promoter and initiator exist and can influence interpretation of tumor progression studies in mouse skin.

Presence of a functionally altered ornithine decarboxylase activity in chrysarobin-promoted mouse epidermal papillomas.

Recent work from this laboratory has demonstrated the presence of a structurally and functionally different ornithine decarboxylase (ODC) in mouse epidermal tumors induced by a two-stage protocol involving initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In this report, the enzymatic properties of ODC present in DMBA-initiated chrysarobin-promoted papillomas are compared to the enzyme induced by chrysarobin in normal epidermis. Analyses of 13 individual tumor extracts indicated each had an elevated level of ODC activity compared to uninduced normal epidermis. Addition of GTP to the enzyme assay caused a marked stimulation of ODC activity in nine of 13 tumor extracts but had no effect on chrysarobin-induced epidermal ODC. Enzyme kinetic analyses indicated that GTP lowered the atypically high apparent Km values for L-ornithine of the papilloma enzyme to values typical of epidermal ODC. The K 1/2 for GTP activation of papilloma ODC was approximately 7 x 10(-9) M. When a series of nucleotides was tested, only GTP, the non-hydrolysable analog GTP gamma S, dGTP and GDP were capable of significant activation at 1 microM, while other derivatives including GMP, ATP and CTP were less effective. The ability of the tumor enzyme to bind GTP was confirmed by the results of GTP-agarose chromatography, in which the papilloma enzyme (but not chrysarobin-induced epidermal ODC) bound to this affinity column and could be eluted by GTP. While some differences were observed in the properties of ODC from chrysarobin-promoted versus TPA-promoted papillomas, the major conclusion of this study is that both agents cause the appearance of a functionally altered ODC in the majority of papillomas produced by a two-step protocol.

Alterations in epidermal polyamine levels and DNA synthesis following topical treatment with chrysarobin in SENCAR mice.

A single topical application of chrysarobin (220 nmol) to SENCAR mouse skin produced alterations in epidermal polyamine levels distinctly different from that following a single topical treatment with 3.4 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). Putrescine and spermidine levels were elevated prior to the induction of epidermal ornithine decarboxylase. In this regard, putrescine levels were elevated at 6 and 24 h after a single application of chrysarobin. In addition, putrescine levels were elevated with a second major peak at 64 h after chrysarobin which coincided with elevated epidermal ornithine decarboxylase activity. Spermidine levels were substantially elevated from 24 to 96 h (peak at 60 h) after a single treatment. TPA treatment produced peak elevations in epidermal putrescine levels at 6 h and epidermal spermidine levels at 24 h after a single treatment. Epidermal spermine levels were dramatically depressed following treatment with chrysarobin (peak depression of approximately 60% below control at 24 h), but only slightly altered following treatment with TPA. The time courses for changes in epidermal DNA synthesis in mouse skin following single treatments with 3.4 nmol of TPA or 220 nmol of chrysarobin also showed considerable differences. TPA treatment produced several waves of DNA synthesis at approximately 18 and 48 h after treatment, while chrysarobin produced a single broad peak at 72 h after treatment. Treatment with chrysarobin was also associated with an initial, dramatic inhibition in epidermal DNA synthesis (to 23% of the control value) which was much more extensive than that elicited by TPA. Inhibition of epidermal DNA synthesis following treatment with chrysarobin was observed within a few hours after treatment and remained depressed until approximately 36 h after treatment. Following treatment with both chrysarobin and TPA, higher levels of epidermal DNA synthesis correlated closely with higher molar ratios of spermidine/spermine, indicating a strong relationship between epidermal spermidine levels and epidermal cell proliferation induced by both promoters. The data suggest that TPA and chrysarobin bring about initial changes in epidermal polyamines by distinct mechanisms; however, both compounds ultimately lead to a dramatic stimulation of epidermal DNA synthesis. These data further support our working hypothesis that anthrones promote skin tumors by an initial mechanism different from that of the phorbol esters.

Modulation of chrysarobin skin tumor promotion.

The present study examined the effect of several prototypic inhibitors of phorbol ester skin tumor promotion on skin tumor promotion by chrysarobin, an anthrone tumor promoter. Retinoic acid (RA) inhibited skin tumor promotion by chrysarobin; however, the degree of inhibition was dependent on the treatment protocol. When RA (10 micrograms/mouse) was given 1 h after each twice-weekly application of chrysarobin (220 nmol/mouse), a marked inhibition of papilloma formation was observed (78%). In additional experiments, using a once-weekly application of chrysarobin, RA also inhibited skin tumor promotion but the magnitude of inhibition was less. Interestingly, RA (10 micrograms/mouse), given 1 or 6 h after the promoter, did not inhibit the induction of epidermal ornithine decarboxylase (ODC) activity induced by a single topical application of chrysarobin (220 nmol). Fluocinolone acetonide (1 microgram/mouse), given 5 min before each twice-weekly application of chrysarobin (220 nmol/mouse) effectively inhibited skin tumor promotion (88%). A 0.5 or 0.25% supplement of alpha-difluoromethylornithine (alpha-DFMO) in the drinking water inhibited the induction of epidermal ODC following chrysarobin (220 nmol/mouse) treatment by 85 or 70%, respectively. Supplements of both 0.25 and 0.5% of alpha-DFMO also led to a 50 and 61% inhibition, respectively, in the number of papillomas per mouse after 25 weeks of promotion with chrysarobin. Interestingly, 0.25% alpha-DFMO in the drinking water did not reduce the number of papillomas per mouse after 20 weeks of promotion with 1.7 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the number of papillomas per mouse that were greater than or equal to 4 mm in diameter was significantly reduced in both chrysarobin- and TPA-treated mice. The data indicate that RA, FA and alpha-DFMO may be general inhibitors of tumor promoter regardless of the chemical class of tumor promoter. The ability of these inhibitors of phorbol ester promotion to inhibit anthrone promotion indicates that some common biochemical pathways may exist for both classes of skin tumor promoters.

Structure-activity relationships for epidermal ornithine decarboxylase induction and skin tumor promotion by anthrones.

The present study was designed to compare the skin tumor promoting and epidermal ornithine decarboxylase (ODC) inducing activities of various structural analogs of anthralin (1,8-dihydroxy-9-anthrone) and chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone). Groups of 30 SENCAR mice each were initiated with 7,12-dimethylbenz[a]anthracene and 2 weeks later promoted with once- or twice-weekly applications of various doses of these anthrone derivatives. Carbon-10 (C10)-acyl derivatives of anthralin were active skin tumor promoters in the range of 25-440 nmol per mouse. 10-Acetylanthralin was significantly more active than 10-myristoyl-anthralin at low doses (e.g. 25 and 50 nmol per mouse) and nearly as potent as the unsubstituted compound. Higher doses (greater than or equal to 100 nmol per mouse) of this derivative were toxic, hence, reducing the final papilloma response. On a relative activity scale where anthralin is 1.0, these derivatives had activities that were approximately 0.7 and 0.2, respectively. 10,10-Dipropylanthralin was totally inactive at the doses tested. C6-Substituted derivatives of chrysarobin demonstrated diverse tumor promoting activities when tested in the range of 25-440 nmol per mouse. On a relative activity scale where chrysarobin is 1.0, 6-methoxychrysarobin (physcion anthrone) was approximately 0.9, whereas 6-hydroxychrysarobin (emodin anthrone) had no activity. Chrysophanic acid (1,8-dihydroxy-3-methyl-9,10-anthraquinone) was also inactive as a tumor promoter at the doses tested. In general, the tumor promoting activities of these anthrone derivatives correlated very well with their ability to induce epidermal ODC after a single topical application indicating an important role for this enzyme in skin tumor promotion by anthones. The ability of C10-substituted derivatives of anthralin to undergo base catalyzed oxidation in vitro correlated with both ODC inducing and tumor promoting activities. In addition, copper(II)bis(diisopropylsalicylate) was found to inhibit both ODC induction and skin tumor promotion by chrysarobin. These latter data, when taken together, suggest a role for oxidation at C10 in skin tumor promotion by anthrone derivatives.

Characterization of skin tumor promotion and progression by chrysarobin in SENCAR mice.

The characteristics of the skin tumor promotion response with anthrone derivatives has been further examined in SENCAR mice. Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) was an effective skin tumor promoter when applied twice weekly with dose-dependent increases in both papillomas and squamous cell carcinomas between 25 and 100 nmol/mouse. A similar dose-response relationship for papilloma and carcinoma formation was observed when chrysarobin was applied once weekly. Interestingly, chrysarobin was approximately twice as active as a skin tumor promoter when applied once weekly versus twice weekly. Doses of 25,100, and 220 nmol/mouse gave maximal papilloma responses of 2.90, 8.15, and 9.38 versus 0.73, 4.70, and 5.42 papillomas/mouse, respectively, in mice initiated with 25 nmol 7,12-dimethylbenz(a)anthracene. Thus, unlike 12-O-tetradecanoylphorbol-13-acetate (TPA), where a twice weekly application frequency is optimal, application of anthrone promoters such as chrysarobin once weekly is a more optimal frequency for papilloma development. Chrysarobin was also a much more effective skin tumor promoter when the start of promotion was delayed by an additional 10 weeks. Thus, groups of mice initiated with 10 nmol 7,12-dimethylbenz(a)anthracene and having promotion started in either the 3rd or the 13th week after initiation had maximal responses of 5.6 or 11.0 papillomas/mouse, respectively. In addition, the rate of papilloma development was faster in the delayed promotion group. The progression of papillomas to carcinomas was examined in all chrysarobin-treated groups and compared with three groups of mice treated with 3.4 nmol TPA. After 60 weeks of promotion, the anthrone promoter-treated groups had carcinoma:papilloma ratios 2.5 to 5.0 times higher than the TPA-treated groups. This was due primarily to the fact that similar carcinoma responses were observed in both anthrone- and TPA-treated mice at optimal promoting doses whereas the papilloma responses were significantly lower in the former groups. The data suggest that anthrone derivatives are very efficient tumor promoters. The results are further discussed in terms of mechanisms of skin tumor promotion.

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.

Kinetics of Haemophilus influenzae type B infection in normal and ribosome-immunized mice using intraperitoneal and intracerebral routes of inoculation.

The kinetics of infection was studied in normal and ribosome-immunized mice challenged with Haemophilus influenzae Type b organisms. Ribosomal preparations extracted by the differential-centrifugation and sodium-dodecyl-sulphate treatment or ammonium-sulphate-precipitation procedures were highly immunoprotective when mice were challenged by the i.p. route. After i.p. injections, organisms rapidly spread to blood, liver, lungs and brain in normal and immunized mice. However, by 24 h after injection, evidence of organism clearance could be seen in immunized mice. By 32 h organisms were cleared from blood, brain and lungs of all immunized mice and from spleens in 2 of 3 mice. However, organisms persisted in high numbers of unimmunized mice until their death by 48 h. These data indicate that i.p. injections of H. influenzae mixed with gastric mucin leads to a true infection and can be used as a model to evaluate immunoprotective activity. The kinetics of infection induced by intracerebral (i.c.) inoculation also was studied. The LD50 for this type of infection was more than 1000 times the LD50 for i.p. infection. The patterns of infection induced by i.c. challenge were similar in normal and immunized mice and immunoprotection could not be detected using this model.