Phorbol ester induction of 8-lipoxygenase in inbred SENCAR (SSIN) but not C57BL/6J mice correlated with hyperplasia, edema, and oxidant generation but not ornithine decarboxylase induction.

Several responses suggested to be critical components of phorbol ester tumor promotion were compared in 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion-sensitive SSIN and TPA promotion-resistant C57BL/6J mice. SSIN mice treated topically with 2 micrograms of TPA showed extensive hyperplasia accompanied by edema, measured as a 26% increase in water content of the skin. Only a very slight hyperplasia and 7% increased water content occurred after TPA treatment of C57BL/6J mice. The induction of ornithine decarboxylase was determined to be the same both in vivo and in vitro for SSIN and C57BL/6J mice, which does not correlate with the histological observations. Because hyperplasia and inflammation can be mediated by arachidonic acid metabolites, it was hypothesized that differences in this metabolic pathway would correlate with the histological responses. No significant qualitative or quantitative differences, however, were observed in the profiles of the major cyclooxygenase products between the strains of mice. Prostaglandin E2, the principal prostaglandin, was synthesized at a 3-fold greater level than prostaglandins D2 or F2 alpha in response to TPA. The most abundant lipoxygenase product was 12-hydroxyeicosatetraenoic acid followed by 8-, 15-, and 5-hydroxyeicosatetraenoic acid. 8-Lipoxygenase activity is elevated 24 h after TPA treatment in the SSIN mice by approximately 4-fold; no elevation is seen in C57BL/6J mice. A comparison of the oxidant response to TPA as well as to phospholipase C showed that SSIN epidermal cells generated a higher level, measured by chemiluminescence, than C57BL/6J cells. This suggests that oxidant generation or possibly 8-lipoxygenase activity may be the basis for the sensitivity or resistance to TPA as a hyperplasiogen and as a tumor promoter.

Correlation of phorbol ester promotion in the resistant C57BL/6J mouse with sustained hyperplasia but not ornithine decarboxylase or protein kinase C.

The activation of protein kinase C, induction of ornithine decarboxylase (ODC), and hyperplasia have been suggested to be linked, sequential processes resulting from phorbol ester application to mouse skin. However, evidence is presented indicating that these events are not necessarily linked or dependent on one another and that significant differences exist in these responses between phorbol ester promotion sensitive (SSIN) and resistant (C57BL/6J) mice. The epidermis from SSIN mice treated with a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) displayed a large induction of ODC and a subsequent extensive hyperplasia. A second TPA treatment at 24 or 48 h after the first did not result in ODC induction (refractory state), and protein kinase C was shown to be down-regulated at these times. By 72 h, however, a responsive state had returned even through protein kinase C remained down-regulated. The epidermis of C57BL/6J responds to a single application of TPA with a level of ODC induction similar to that of the SSIN mice. Protein kinase C was down-regulated by approximately 75% after 24 h and was virtually completely down-regulated at 48 and 72 h (95-97%). In contrast to the above findings for the sensitive mice, however, little, if any, hyperplasia was produced. In addition, while a second TPA treatment at 24 h did not result in ODC induction (refractory state), hyperplasia did occur within 24 to 48 h. When the second TPA application was given 48 h after the first, at a time when protein kinase C was down-regulated, an overinduction of ODC occurred, as well as subsequent hyperplasia. Furthermore, a significant number of papillomas resulted when these increased treatment frequencies, i.e., once a day or every other day, were used to promote dimethylbenz(a)anthracene-initiated C57BL/6J mice. It is concluded that, while hyperplasia remains an apparent requirement for tumor promotion, the ODC induction following an initial TPA treatment is insufficient for or not causally related to this hyperplasia. In addition, subsequent ODC induction, at least in the C57BL/6J mouse, is probably not mediated by protein kinase C.

Arachidonic acid metabolism varies with the state of differentiation in density gradient-separated mouse epidermal cells.

Epidermal cells were isolated from adult inbred SENCAR (SSIN) mice and separated by density-gradient centrifugation. The cells were pooled into three fractions shown by previous work to differ in their state of differentiation and proliferative potential. The three fractions were examined for their capacity to metabolize exogenous 14C-arachidonic acid (AA) into prostaglandins (PG) and hydroxyeicosatetraenoic acids (HETE). Cells found in the upper two fractions, which are less dense, have less proliferative potential in vitro, and are more differentiated than cells in the lower more dense fraction, are much more active in producing PG from exogenous AA than are the more dense cells. This was observed in intact cells as well as cells disrupted by freeze-thawing following density separation. The same relationship was found for HETE production in that cytoplasmic preparations from the two fractions containing the less dense cells were much more active in the production of HETE than cytoplasmic preparations from the more dense fraction. The two upper fractions differed little from each other in the production of PG or HETE. These results indicate the presence of higher levels of active cyclooxygenase and lipoxygenases in fractions containing the less dense, more differentiated cells than in the fraction containing the more dense, less differentiated cells which are highly enriched for basal keratinocytes.

Mediation of phorbol ester-induced oxidant generation in murine epidermal cells by protein kinase C.

Murine epidermal cells have previously been shown to produce an oxidant response to the mouse skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA); however, the cellular source of these oxidants has not been well characterized. The demonstration that phospholipase C also elicits this oxidant response suggests that protein kinase C is the common mediator. In order to pursue this hypothesis, studies using protein kinase C inhibitors were carried out; both the induced oxidant response and the induction of ornithine decarboxylase (ODC), a known protein kinase C mediated event, were studied. At 100 µm-palmitoylcarnitine inhibited TPA-induced ODC by 50% and phospholipase C-induced ODC by 95%. At the same dose level, the negative analogue acetylcarnitine had no inhibitory effect on ODC with either inducer. The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7) and its negative analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide hydrochloride (HA-1004) were also used. At 100 µm, H-7 completely inhibited both TPA and phospholipase C-induced ODC; at the same dose HA-1004 had no effect. When these agents were included in the chemiluminescence assay for oxidant generation, similar results were seen: at 100 µm-palmitoylcarnitine and H-7, but not acetylcarnitine or HA-1004, suppressed the response by nearly 100%. These results suggest that the oxidant response to TPA in epidermal cells is mediated at least in part by protein kinase C.

Reactive oxygen in the tumor promotion stage of skin carcinogenesis.

Exposure of isolated SENCAR mouse epidermal cells to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) in vitro resulted in the production of oxidant species detected as chemiluminescence. This oxidant response can be inhibited by superoxide dismutase and copper complexes but not catalase or scavengers of hydroxyl radical or singlet oxygen, suggesting that the oxidant is superoxide anion. Inhibitors of various parts of the arachidonate cascade affect the TPA-induced oxidant response in a manner that corresponds to their effects on in vivo tumor promotion experiments. Agents that inhibit lipoxygenase activity, i.e. nordihydroguaiaretic acid, benoxaprofen, but not agents that are cyclooxygenase inhibitors, i.e. indomethacin, are effective in suppressing the oxidant response to TPA. Phospholipase C but not phospholipase A2 or D produced an oxidant response kinetically similar to that elicited by TPA. The inhibitors of TPA-induced oxidants inhibited the phospholipase C response to the same extent, suggesting that TPA and phospholipase C may produce an oxidant species through a common mechanism, via phospholipid turnover-protein kinase C activation. The relevance of oxidant production to the tumor promotion process is suggested by the ability of exogenous xanthine/xanthine oxidase, a superoxide anion-generating system, to induce ornithine decarboxylase, a characteristic of TPA-treated cells. In addition, oxidant production is significantly lower in cells from the TPA-promotion resistant C57BL/6J mouse. These studies provide further support for a role for reactive oxygens in the tumor promotion process.

A simple high-performance liquid chromatographic method for the measurement of 2-methylsulfonylpyridine in plasma.

A simple and sensitive high-performance liquid chromatographic (HPLC) method for the analysis of 2-methylsulfonylpyridine (2-MSP) in plasma has been developed. Up to 1 mL of plasma containing 2-MSP and an internal standard was extracted with 3 mL of methylene chloride, usually twice, evaporated to dryness, resuspended in mobile phase, and chromatographed on two 15-cm C8 reversed-phase LC columns in series. The mobile phase was 5% acetonitrile in water with a flow rate of 1 mL/min and ultraviolet detection was at 260 nm. Extraction of plasma produced no interfering endogenous components and the recovery of 2-MSP was 60-70%. The intraday and interday statistics of 2-MSP standard curves from 10 to 320 ng in plasma demonstrate that the assay is sensitive and precise using either human or monkey plasma and any plasma volume up to 1 mL.

Pharmacokinetics of topically applied ciprofloxacin in rabbit tears.

Fluoroquinolones provide an important single antibiotic therapy for bacterial keratitis caused by Staphylococcus aureus and numerous gram-negative bacteria, including Pseudomonas aeruginosa. The pharmacokinetics of three ocular fluoroquinolones, norfloxacin (CHIBOXIN, Merck, Sharp & Dohme), ciprofloxacin (CILOXAN, Alcon Laboratories), and ofloxacin (OCUFLOX, Allergan Pharmaceuticals), have been studied in the human eye and in both the normal rabbit eye and rabbit models of keratitis. However, the pharmacokinetics of ciprofloxacin have not been previously studied in the tear film of rabbits. This study was done to determine the pharmacokinetics of topical ciprofloxacin in the rabbit tear film. Two drops of CILOXAN were applied to the eyes of normal rabbits. Tear samples were collected at 5, 10 and 30 minutes, and 1, 2, 4 and 6 hours after topical drug application. Tear samples were analyzed for ciprofloxacin concentrations by HPLC. Ciprofloxacin concentrations reached a peak at 5 minutes, then declined in a manner similar to that reported for norfloxacin and ofloxacin. The ciprofloxacin concentrations in tears were substantially higher throughout the length of the study than the MIC90 for most ocular pathogens including Staphylococcus aureus and Pseudomonas aeruginosa.

Possible dissociation of the phorbol ester-induced oxidant response and tumor promotion in the F1 offspring of SSIN x C57BL/6J mice.

The F1 progeny of a cross between 12-O-tetradecanoyl-phorbol-13-acetate (TPA) tumor promotion-sensitive SSIN mice and TPA promotion-resistant C57BL/6J mice were found to be sensitive to TPA as a tumor promoter. The tumor response was substantial, with an average of 15 papillomas per mouse and a 100% incidence following initiation with 400 nmol dimethylbenz[a]anthracene and promotion with 6.5 nmol (4 micrograms) TPA. To determine whether tumor promotability of the F1 mice correlates with other parameters believed to be associated with TPA responsiveness, oxidant generation, epidermal hyperplasia and edema were compared in the parents and F1 hybrids. The SSIN produced a strong hyperplastic response to TPA, the C57BL/6J a negligible response and the F1 hybrids a moderate response. In the SSIN, 6.5 nmol (4 micrograms) TPA caused an 18% increase in the water content of the skin (edema) while no change was seen for either the C57BL/6J or F1 hybrids. The oxidant response of the F1 hybrids to either TPA or phospholipase C was markedly less than that observed for the SSIN and was similar to the response previously observed for the C57BL/6J mice. These findings suggest that the oxidant response may not be an essential aspect of TPA tumor promotion. It may be related to the edema response, suggesting that at least this aspect of inflammation is not necessary for promotion.

Binding of benzo[a]pyrene and intracellular transport of a bound electrophilic benzo[a]pyrene metabolite by lipoproteins.

Human serum lipoproteins were isolated by means of size exclusion h.p.l.c. Non-covalent uptake of [3H]benzo[a]pyrene was quantitated for fractions collected from the effluent of a liquid chromatographic separation of human serum, and was found to directly correlate with the lipoprotein concentration. An electrophilic benzo[a]pyrene metabolite, [3H]trans 7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene, non-covalently associated with low density lipoproteins was transferred to human lymphocytes in vitro and bound acid-precipitable nucleic acids of the lymphocytes as a function of time. Benzo[a]pyrene metabolite binding to lymphocyte DNA was demonstrated by means of CsCl density gradient analysis. Non-mitogen-stimulated lymphocytes exposed to very low concentrations of carcinogen in the presence of low density lipoprotein demonstrated [3H]thymidine incorporation; without the concomitant addition of low density lipoprotein the low concentrations of carcinogen did not stimulate [3H]thymidine incorporation.

Separation and detection of lipoproteins in human serum by use of size-exclusion liquid chromatography: a preliminary report.

Components of human serum can be separated on the basis of differences in relative molecular mass by using size-exclusion "high-performance" liquid chromatography. Lipoproteins in fractions of the eluate can be quantitated by conventional chemical and enzymatic methods. Alternatively, if lipoproteins in the serum are selectively prestained with diformazan dye, the column effluent can be monitored spectrophotometrically at 580 nm, so that only the lipoprotein components of serum are detected. Samples of purified low-density lipoproteins, so stained and analyzed, provide peak-area values that are proportional to their concentration as evaluated by chemical methods. With this technique, the various lipoprotein classes can be quickly separated and their concentration estimated. These techniques should prove useful in clinical and research laboratories.