Genomic profiling of late-onset basal cell carcinomas from two brothers with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant genetic disorder. It is commonly caused by mutations in PTCH1 and chiefly characterized by multiple basal cell carcinomas (BCCs) developing prior to the age of 30 years. In rare cases, NBCCS presents with a late onset of BCC development. OBJECTIVE: To investigate BCC tumorigenesis in two brothers, who showed characteristic features of NBCCS but developed their first BCCs only after the age of 40 years. Two other siblings did not show signs of NBCCS. RESULTS: We obtained blood samples from four siblings and nine BCCs from the two brothers with NBCCS. Whole exome sequencing and RNA sequencing revealed loss of heterozygosity (LOH) of PTCH1 in eight out of nine tumours that consistently involved the same haplotype on chromosome 9. This haplotype contained a germinal splice site mutation in PTCH1 (NM_001083605:exon9:c.763-6C>A). Analysis of germline DNA confirmed segregation of this mutation with the disease. All BCCs harboured additional somatic loss-of-function (LoF) mutations in the remaining PTCH1 allele which are not typically seen in other cases of NBCCS. This suggests a hypomorphic nature of the germinal PTCH1 mutation in this family. Furthermore, all BCCs had a similar tumour mutational burden compared to BCCs of unrelated NBCCS patients while harbouring a higher number of damaging PTCH1 mutations. CONCLUSIONS: Our data suggest that a sequence of three genetic hits leads to the late development of BCCs in two brothers with NBCCS: a hypomorphic germline mutation, followed by somatic LOH and additional mutations that complete PTCH1 inactivation. These genetic events are in line with the late occurrence of the first BCC and with the higher number of damaging PTCH1 mutations compared to usual cases of NBCCS.

Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.

BACKGROUND: Interleukin (IL)-31 is a novel Th2 T-cell cytokine that induces pruritus and dermatitis in transgenic mice. While enhanced mRNA expression of this cytokine is detected in skin samples of inflammatory skin diseases, the regulation of IL-31 expression is poorly understood. OBJECTIVES: To assess the effects of ultraviolet (UV) B radiation and H2O2 on IL-31 mRNA and protein expression in skin and different peripheral blood mononuclear cells (PBMCs). METHODS: The effects of UVB radiation and H2O2, as a prototypic reactive oxygen species, on IL-31 mRNA and protein expression were analysed in various inflammation-related cells and murine skin tissue. RESULTSTreatment of cells with UVB radiation and H2 O2 strongly induced IL-31 mRNA and protein expression in human PBMCs and in the skin of SKH-1 mice. Following exposure to UVB or H2O2, we observed increased expression of IL-31 mRNA in T cells, monocytes, macrophages, and immature and especially mature dendritic cells. H2O2 treatment but not UVB radiation led to a moderate upregulation of IL-31 mRNA expression in epidermal keratinocytes and dermal fibroblasts. Pretreatment of T lymphocytes with the MAPK p38 inhibitor SB203580 or the MEK1 inhibitor U0126 reduced the stimulatory effect of H2O2. These experiments suggest that p38 is involved in the regulation of IL-31 expression in human skin. CONCLUSIONS: Our studies reveal that UVB and reactive oxygen species stimulate the expression of IL-31 in PBMCs and skin, especially in T cells, monocytes and monocyte-derived dendritic cells.

Expression and function of cytochrome p450-dependent enzymes in human skin cells.

Scientific interest in defining the human body's ability to limit the effects of administered drugs and xenobiotics dates from the mid-19th century when developing knowledge and techniques in the field of organic chemistry first made such studies possible. The first experimental evidence documenting the existence of cytochrome p450 (CYP) dates to the year 1955, when an enzyme system capable of oxidizing xenobiotic compounds was identified in the endoplasmic reticulum of liver homogenates. From these days on several studies analyzed the expression and function of metabolizing phase I enzymes in liver cells. Due to the unique structural features of human skin, little was known about the expression and function of CYP enzymes in this tissue and their role in uptake, metabolism and elimination of xenobiotics as well as endogenous substrates. Lasting recent years it has become clear that human skin cells express various CYP enzymes, including CYP26AI which is responsible for the metabolism of retinoic acid in skin cells. It has been also shown that CYP enzyme expression patterns are cell type and tissue specific and that in skin cells this differs significantly from its expression in other environmental interfaces such as the liver, lung and gastrointestinal tract. Therefore knowledge of skin-specific CYP expression and function is a prerequisite for pharmacological studies of the skin.

Human skin aryl hydrocarbon hydroxylase. Induction by coal tar.

Coal tar products, which are widely used in treating dermatologic disease, contain numerous polycyclic aromatic hydrocarbons, including 3,4-benzo[a]pyrene (BP). BP is among the most potent environmental chemical carcinogens and is known to evoke tumors in the skin of experimental animals and perhaps also of man. In this study the effect of cutaneous application of coal tar solution (U. S. Pharmacopeia) on aryl hydrocarbon hydroxylase (AHH) activity in the skin of patients usually treated with this drug was investigated. AHH, a cytochrome P-450 dependent carcinogen-metabolizing enzyme appears to play an important role in the activation of polycyclic hydrocarbons into reactive moieties that can bind to DNA and that may directly induce cancer. Application of coal tar solution to human skin caused a two to five-fold induction of cutaneous AHH in nine subjects. In further studies, the incubation of human skin with coal tar solution in vitro also caused variable induction of cutaneous AHH. Maximum responses in both systems occurred after 24 h and enzyme activity in vitro was time- and tissue- and substrate-concentration dependent. Studies in experimental animals showed that topical application of coal tar solution caused induction of AHH in skin and, after percutaneous absorption, in liver as well. Assay of several defined constituents of coal tar for AHH induction showed that BP was the most potent inducer of AHH tested. These studies indicate that topical application of coal tar solution in doses ordinarily used in treating dermatologic disease causes induction of AHH in human skin and suggest that such induced enzymatic activity could relate to carcinogenic responses to this agent in skin or, after percutaneous absorption, in other tissues as well.

Uroporphyrin I stimulation of collagen biosynthesis in human skin fibroblasts. A unique dark effect of porphyrin.

Porphyria cutanea tarda and erythropoietic porphyria are disorders of heme synthesis that originate in the liver and bone marrow, respectively. Each is characterized by increased accumulation of uroporphyrin, I, by cutaneous photosensitivity, and in some patients by indurated plaques and scarring that resemble scleroderma. These scleroderma-like lesions occur in light-exposed and light-protected body areas. In these studies we evaluated the role of uroporphyrin I and of light in evoking the scleroderma-like cutaneous changes. Normal human skin fibroblasts were exposed to uroporphyrin I and to 400 nm radiation and the effect of these agents on collagen accumulation by the cells was determined. Radioactive tracer studies showed that uroporphyrin I caused a specific increase in the accumulation of newly synthesized collagen by fibroblast monolayer cultures, as verified by [(3)H]hydroxyproline and collagenase digestion assays. Collagen accumulation was stimulated 1.5- to 2.7-fold by uroporphyrin I, whereas noncollagenous protein accumulation was unchanged. The increased collagen accumulation was time and uroporphyrin I-concentration-dependent, and occurred both in the presence or absence of ultraviolet light exposure. Further studies demonstrated that the increased accumulation was not the result of decreased rates of collagen degradation nor was it due to changes in cell population growth parameters (generation times and saturation densities). No changes in morphology of the treated cells occurred. These studies indicate that porphyrins possess previously undemonstrated biological effects that are independent of their photosensitizing properties. This novel dark effect of uroporphyrin I may account for the sclerodermatous lesions seen in the skin of patients with porphyria cutanea tarda and erythropoietic porphyria.

A novel mechanism for the generation of superoxide anions in hematoporphyrin derivative-mediated cutaneous photosensitization. Activation of the xanthine oxidase pathway.

Prior studies, both in vitro and in vivo, have suggested that cutaneous porphyrin photosensitization requires the generation of superoxide anion (.O2-) and various other reactive oxygen metabolites. No unifying concept has emerged, however, that unequivocally demonstrates the source of generation of these species. Since xanthine oxidase is known to generate .O2- in reperfused ischemic tissue and in certain inflammatory disorders, we attempted to assess its role in porphyrin photosensitization. C3H mice were rendered photosensitive by the intraperitoneal administration of dihematoporphyrin ether (DHE) (5 mg/kg) followed by irradiation with visible light. Murine ear swelling was used as a marker of the acute photosensitization response and involvement of oxygen radicals was evaluated using electron spin resonance (ESR) spectroscopy. The administration of allopurinol, a potent inhibitor of xanthine oxidase, afforded 90% protection against DHE-mediated acute photosensitivity in vivo. Furthermore, xanthine oxidase activity was twofold higher in the skin of photosensitized mice than in unirradiated animals. ESR spectra of 5,5-dimethyl-1-pyrroline N-oxide-trapped radicals from the skin of photosensitized mice verified the presence of .O2- and .OH, while neither of these species was detected in the skin of control mice or mice receiving allopurinol. The administration of a soybean trypsin inhibitor or verapamil before irradiation also partially blocked the photosensitivity response, suggesting that calcium-dependent proteases play a role in the activation of xanthine oxidase in this photodynamic process. These data provide in vivo evidence for the involvement of .O2- in DHE-mediated cutaneous photosensitization and suggest that these radicals are generated through the activation of the xanthine oxidase pathway. The administration of allopurinol and calcium channel blockers may thus offer new approaches for the treatment of cutaneous porphyrin photosensitization.

Induction of a deficiency of steroid delta 4-5 alpha-reductase activity in liver by a porphyrinogenic drug.

The hepatic enzymes that catalyze drug oxidations and the reductive metabolism of steroid hormones to 5alpha-derivatives are localized in membranes of the endoplasmic reticulum. Phenobarbital, which exacerbates acute intermittent porphyria in man, induces drug-oxidizing enzymes in liver. Additionally, patients in whome the primary gene defect (uroporphyrinogen-I-synthetase deficiency) of acute intermittent porphyria has become clinically expressed have low levels of hepatic steroid delta4-5alpha-reductase activity. This 5alpha-reductase deficiency in acute intermittent porphyria leads to the disproportionate generation of 5beta-steroid metabolites from precursor hormones; such steroid metabolites have significant porphyria-inducing action experimentally. In this study the effects of phenobarbital on drug oxidation and steroid 5alpha-reduction in man were examined to determine if this drug could produce changes in steroid 5alpha-reductase activity which mimicked those seen in patients with acute intermittent porphyria. Metabolic studies with [14C]-testosterone and 11beta-[3H]hydroxyandrostenedione were carried out in five normal volunteers. In all five subjects phenobarbital administration (2 mg/kg/per day for 21 days) enhanced plasma removal of the test drugs antipyrine and phenylbutazone as expected; but in four subjects phenobarbital also substantially depressed 5alpha-metabolite formation from [14C]testosterone and resulted in a pattern of hormone biotransformation characterized by a high ratio of 5beta/5alpha-metabolite formation. Studies with 11beta-[3H]hydroxy-androstenedione in three subjects confirmed that phenobarbital produced this high 5beta/5alpha ratio of steroid metabolism by depressing 5alpha-reductase activity for steroid hormones in liver. The high ratio of 5beta/5alpha-metabolites formed in normals after drug treatment mimicks the high 5beta/5alpha-steroid metabolite ratio formed from endogenous hormones in acute intermittent porphyria. The proximate mechanism by which phenobarbital induces reciprocal changes in activities of the microsomal enzymes which catalyze drug oxidations and steroid 5alpha-reductions is not known. This action of phenobarbital raises the possibility, however, that certain drugs which provoke exacerbations of human porphyria may do so, in part, by producing deleterious shifts in the patterns of endogenous steroid hormone metabolism.

Inhibition of 3-methylcholanthrene-induced skin tumorigenicity in BALB/c mice by chronic oral feeding of trace amounts of ellagic acid in drinking water.

Chronic p.o. feeding of small amounts of ellagic acid, a naturally occurring dietary plant phenol, to BALB/c mice in drinking water afforded significant protection against skin tumorigenesis induced by 3-methylcholanthrene, a polycyclic aromatic hydrocarbon carcinogen. A significant increase in the latent period for the development of skin tumors by 3-methylcholanthrene was observed in the ellagic acid-fed group of mice (9 wk on test) as compared to the control group of animals (6 wk on test). The observed protection against tumor induction in the ellagic acid-fed group of animals may be due to the inhibition of the metabolic activation of the polycyclic aromatic hydrocarbon since epidermal aryl hydrocarbon hydroxylase activity was found to be significantly inhibited. Our results suggest that dietary supplementation with small amounts of ellagic acid may prove useful in reducing the risk of skin carcinogenesis induced by environmental chemicals.

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats.

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H2O2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.

Inhibition of polycyclic aromatic hydrocarbon-DNA adduct formation in epidermis and lungs of SENCAR mice by naturally occurring plant phenols.

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid are known to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs). The binding of bay-region diol-epoxides of PAHs to target tissue DNA is thought to be essential for the initiation of cancer by these compounds. In this study we investigated the effect of these plant phenols on PAH-DNA adduct formation in the epidermis and lung of SENCAR mice. In vitro addition of tannic acid, quercetin, myricetin, and anthraflavic acid (25 microM) to an incubation system containing epidermal microsomes prepared from either control or 3-methylcholanthrene-pretreated mice inhibited benzo(a)pyrene binding to calf thymus DNA by 63-64, 38-43, 36-37, and 27-33%, respectively. A single topical application of tannic acid, quercetin, myricetin, and anthraflavic acid at a dose of 400 mumol/kg body weight resulted in the inhibition of [3H]benzo(a)pyrene binding to epidermal DNA (48-73%) and protein (51-63%). The same dose of these plant phenols (400 mumol/kg) caused even greater inhibition of (+/-)-[3H]-7 beta,8 alpha-dihydroxy-7,8-dihydrobenzo(a)pyrene and [3H]-7,12-dimethybenz(a)anthracene binding to epidermal DNA and protein. The formation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-deoxyguanosine adducts was substantially diminished in both epidermis (62-86%) and lungs (38-84%). These results indicate that tannic acid, quercetin, myricetin, and anthraflavic acid are potent inhibitors of carcinogen binding to epidermal and lung DNA and suggest that these plant phenols could prove useful in modifying the risk of tumor induction by PAHs such as benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in these two tissues.

Immunogenetic influences on the initiation stage of the cutaneous chemical carcinogenesis pathway.

While it is generally agreed that environmental exposure to solar radiation and to certain classes of chemicals are the major causes of nonmelanoma skin cancer, it is also believed that genetic polymorphisms regulating immunological responses are important determinants of individual susceptibility to skin cancer. However, little is known about their interactions with the chemical carcinogenesis pathway prior to the actual development of tumors. This issue was examined by comparing susceptibility to skin cancer in C3H/HeN and C3H/HeJ mice, two strains that differ only at the lipopolysaccharide genetic locus, which serves as a regulator of a number of immunological activities. When subjected to a two-stage cutaneous tumorigenesis protocol, C3H/HeJ mice, which have a mutation at the lipopolysaccharide genetic locus that renders them deficient in their capacity to produce cytokines and to activate macrophages, developed nearly three times as many tumors as did C3H/HeN mice, which do not have this mutation. Epidermal DNA binding of 7,12-[3H]dimethylbenz(alpha)anthracene, an index of tumor initiation, was also significantly greater in C3H/HeJ than in C3H/HeN mice. Immunological activities regulated by the lipopolysaccharide genetic locus thus confer resistance to DMBA-induced cutaneous tumorigenesis in mice and are associated with changes that occur early in the tumorigenesis pathway, prior to the development of tumors.

Inhibition of epidermal xenobiotic metabolism in SENCAR mice by naturally occurring plant phenols.

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid have been shown to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocarbons including benzo(a)pyrene (BP). The present study was designed to determine whether these plant phenols can alter epidermal cytochrome P-450-dependent monooxygenases in SENCAR mice. In vitro addition of these plant phenols to epidermal microsomal preparations inhibited aryl hydrocarbon hydroxylase (AHH) activity in a concentration-dependent manner. The 50% inhibitory concentrations for tannic acid, myricetin, quercetin, and anthraflavic acid ranged from 4.4 X 10(-5) M to 12.4 X 10(-5) M in microsomes prepared from control and 3-methylcholanthrene-pretreated animals. Of the plant phenols studied tannic acid was found to be the most potent inhibitor of epidermal AHH activity. Tannic acid, quercetin, myricetin, and anthraflavic acid exhibited a mixed type of inhibitory effect with Ki values of 81, 63, 135, and 165 microM, respectively. In vitro addition of these plant phenols (240 microM) to the incubation mixture prepared from control and 3-methylcholanthrene-treated animals resulted in varying degrees of inhibition of epidermal microsomal AHH (57-92%), ethoxycoumarin O-deethylase (19-58%), and ethoxyresorufin O-deethylase (33-85%) activities. High pressure liquid chromatographic analysis of the organic solvent-soluble metabolites of BP produced by epidermal microsomes indicated a substantial decrease in the formation of BP-diols (23-67%) and BP-phenols (29-57%) by each of the plant phenols. The formation of BP-7,8-diol was substantially inhibited (29-52%) by each of the plant phenols. Further in vivo studies showed that a single topical application of tannic acid, quercetin, and myricetin greatly diminished epidermal AHH (53-65%), ethoxycoumarin O-deethylase (30-68%), and ethoxyresorufin O-deethylase (66-97%) activities whereas anthraflavic acid was ineffective in this regard even when repeatedly applied. Our results indicate that plant phenols have substantial though variable inhibitory effects on epidermal monooxygenase activities and BP metabolism suggesting that these compounds may be capable of inhibiting the carcinogenic effects of polycyclic aromatic hydrocarbons in the skin.

Photoenhancement of lipid peroxidation associated with the generation of reactive oxygen species in hepatic microsomes of hematoporphyrin derivative-treated rats.

Hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative (HPD) undergo rapid enhancement of lipid peroxidation in the presence of solar radiation (approximately 400 nm). Quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, and inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethanol, largely protected against the enhancement of lipid peroxidation caused by HPD photosensitization. Catalase, a scavenger of hydrogen peroxide and superoxide dismutase, a scavenger of superoxide anion, had little or no protective effect against HPD-photosensitized enhancement of lipid peroxidation. Our data indicate that in vitro irradiation of hepatic microsomes prepared from HPD-treated rats results in the generation of both singlet oxygen and hydroxyl radical. These reactive moities are associated with a rapid increase in microsomal lipid peroxidation which may explain the unique susceptibility of membranous components of cells to this type of phototoxic injury.

Exceptional activity of tannic acid among naturally occurring plant phenols in protecting against 7,12-dimethylbenz(a)anthracene-, benzo(a)pyrene-, 3-methylcholanthrene-, and N-methyl-N-nitrosourea-induced skin tumorigenesis in mice.

Our recent studies have shown that naturally occurring dietary plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid are capable of inhibiting polycyclic aromatic hydrocarbon (PAH) metabolism and subsequent PAH-DNA adduct formation in epidermis of SENCAR mice (M. Das, et al., Cancer Res., 47: 760-766, 1987, and 47: 767-773, 1987). In this study these plant phenols were tested for their effects against PAHs and N-methyl-N-nitrosourea-induced skin tumorigenesis in mice. Each plant phenol was evaluated as a possible anticarcinogen in an initiation and promotion and a complete skin tumorigenesis protocol. In the two-stage tumor protocol in SENCAR mice using 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N-nitrosourea as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate as tumor promoter each plant phenol afforded significant protection against skin tumorigenicity. The protective effects were verified both by prolongation of latency period and by subsequent tumor development. In the complete carcinogenesis protocol in BALB/c mice using 3-methylcholanthrene as a tumorigen the applications of each of the plant phenols 30 min prior to each PAH application afforded significant protection by delaying the onset and the subsequent development of skin tumors. Our results suggest that these plant phenols have substantial though variable potential for modifying the risk of skin tumorigenicity induced by a wide variety of chemicals and of these tannic acid was shown to have maximal chemoprotective effects.

Clotrimazole, an inhibitor of epidermal benzo(a)pyrene metabolism and DNA binding and carcinogenicity of the hydrocarbon.

Clotrimazole, a topically applied imidazole antifungal agent widely used in dermatological practice, was shown to be a potent inhibitor of the epidermal metabolism of benzo(a)pyrene (BP) and its microsomal enzyme-mediated binding both to neonatal rat epidermal DNA in vivo and to calf thymus DNA in vitro. Varying concentrations of clotrimazole added to in vitro incubation systems resulted in a dose-dependent inhibition of cytochrome P-450-dependent microsomal aryl hydrocarbon hydroxylase (AHH) in control animals as well as in animals pretreated with topical application of known inducers of the enzyme. Inhibition of epidermal AHH by topically applied clotrimazole was time and dose dependent. The 50% inhibition of clotrimazole for epidermal AHH ranged from 0.12 to 0.25 microM, which suggests that clotrimazole is among the most potent inhibitors of epidermal AHH yet identified. Clotrimazole was also found to be a potent inhibitor of epoxide hydrolase activity in vitro with a 50% inhibition at 0.1 mM. High-pressure liquid chromatographic analysis of the metabolism of BP in rat epidermal microsomes revealed substantial inhibition of metabolite formation by clotrimazole. This occurred in microsomes prepared from untreated as well as animals pretreated with inducers of the enzyme. Furthermore, a single topical application of clotrimazole resulted in 80 and 30% induction of epidermal and hepatic glutathione S-transferase activity, respectively. Topical application of clotrimazole to the skin of BALB/c mice substantially increased the latent period for the development of skin tumors by 3-methylcholanthrene. These studies indicate that clotrimazole is an extremely potent inhibitor of epidermal BP metabolism and of the DNA-binding of polycyclic aromatic hydrocarbon (PAH) carcinogens, and is an enhancer of enzymes necessary for detoxification of the PAH. Clotrimazole also reduces the formation of carcinogenic and mutagenic metabolites of BP in vitro and in vivo and inhibits induction of skin tumors by the PAH. These data indicate that the imidazole antifungal clotrimazole offers promise as an agent useful for the modulation of PAH cancer risk in the skin.

Enhancement of bleomycin-mediated DNA damage by epidermal microsomal enzymes.

The role of epidermal microsomal enzymes in catalyzing bleomycin-mediated chain breakage in calf-thymus DNA and in DNA isolated from neonatal rat epidermis was studied. Aerobic incubation of bleomycin with epidermal microsomes, epidermal or calf-thymus DNA and NADPH caused substantial chain breakage of the DNA which was dependent upon concentrations of drug, microsomal protein and NADPH. The reactive oxygen scavenger superoxide dismutase, the metal chelator EDTA, and cytochrome c each inhibited the enzyme-mediated chain breakage reaction. Scavengers of hydrogen peroxide and hydroxyl radicals, including catalase and benzoate and inhibitors of microsomal cytochrome P-450-dependent monooxygenases such as 1-benzylimidazole, metyrapone and alpha-naphthoflavone, had no inhibitory effects on bleomycin-mediated DNA chain breakage. In contrast, ascorbic acid significantly enhanced DNA damage by bleomycin. These studies indicate that mammalian epidermis possesses membrane-bound enzyme activity capable of enhancing bleomycin-mediated chain breakage of DNA and that oxidation/reduction of adventitious iron and generation of reactive oxygen participate in the reaction. These responses in the epidermis could directly relate to the mechanism of action of intralesional injections of bleomycin which are used quite effectively in the management of recalcitrant human warts. Either epidermal or wart virus DNA or both could be targets for this pharmacologic effect of the drug which is augmented by epidermal microsomal enzymes.

Treatment of the porphyrias: mechanisms of action.

The porphyrias are diseases that result from inherited or acquired abnormalities of porphyrin-heme synthesis in the liver and the bone marrow. Only the hepatic porphyrias are known to be aggravated by exposure to a variety of exogenous drugs and chemicals. Simple avoidance of these agents will reduce the risk of developing hepatic porphyria and may lead to clinical improvement in patients with active disease. Some types of therapy of the hepatic porphyrias are effective because of their ability to modulate the activity of delta-aminolevulinic acid synthetase, the rate-limiting enzyme for heme synthesis. Most of the porphyrias are associated with cutaneous photosensitivity, the treatment of which centers about either reducing the excessive production of porphyrins or of inhibiting the photobiological response to these photosensitizing chemicals in the skin.

Differential role of reactive oxygen intermediates in photofrin-I- and photofrin-II-mediated photoenhancement of lipid peroxidation in epidermal microsomal membranes.

Photoradiation therapy with porphyrins and light offers an alternative approach to the management of certain types of cancer. The mechanism of tissue destruction mediated by this modality is poorly understood. In this study, epidermal microsomes incubated in vitro with Photofrin-I (Pf-I) and Photofrin-II (Pf-II) followed by exposure to radiation (approximately 400 nm) resulted in increased (180%) NADPH-supported (enzymatic) as well as ADP/iron-supported (140%) (nonenzymatic) lipid peroxidative damage as measured by malondialdehyde formation. Lipid peroxidation by Pf-I and Pf-II was found to be differentially affected by quenchers of singlet oxygen (2,5-dimethylfuran, histidine, beta-carotene, ascorbic acid, and sodium azide), superoxide anion (superoxide dismutase), and the hydroxyl radical (sodium benzoate, mannitol, and ethanol). Catalase, a quencher of hydrogen peroxide, afforded significant protection only against Pf-II-enhanced lipid peroxidative damage while it had little effect against the Pf-I-mediated reaction. Deuterium oxide, which is known to increase the half-life of singlet oxygen, was found to enhance Pf-I-mediated lipid peroxidation but produced insignificant effects upon Pf-II-mediated photosensitization. Our results indicate that Pf-I and Pf-II, which are employed for the photodynamic therapy of malignant tumors, evoke membrane damage by generating different reactive oxygen species. The Pf-I-mediated photodestruction mainly involves a type II mechanism via singlet oxygen formation, whereas Pf-II-mediated photodestruction preferentially involves a type I mechanism by generating superoxide anions and hydroxyl radicals. Our data indicate that tumor necrosis evoked by porphyrins and light is likely due to the generation of reactive oxygen species.

Studies on the role of reactive oxygen species in mediating lipid peroxide formation in epidermal microsomes of rat skin.

The role of superoxide, hydrogen peroxide, and singlet oxygen in mediating nonenzymic and NADPH-supported enzymic lipid peroxidation in skin microsomes was investigated. Incubation of skin microsomes with NADPH and/or Fe+3-ADP or ascorbate resulted in the formation of lipid peroxides. The epidermis was the major target site for microsomal lipid peroxide formation in skin. Enzymic peroxidation of epidermal microsomes required NADPH and was oxygen-dependent. Addition of the nonenzymic catalysts, Fe+3 and ADP, to the enzymic peroxidation system had an additive effect on the generation of lipid peroxide in epidermal microsomes. Epidermal microsomal lipid peroxidation was inhibited by singlet oxygen quenchers such as dimethylfuran, histidine, and beta-carotene. Hydroxyl ion scavengers such as mannitol, benzoate, or ethyl alcohol and the enzymic scavenger of superoxide, superoxide dismutase, were all ineffective in this respect. Addition of EDTA, Mn+2, cytochrome c+3, and catalase to the NADPH-supported enzymic peroxidation system resulted in strong inhibition of lipid peroxide formation in skin. Glutathione or epidermal cytosol added alone or in combination to the NADPH-supported incubation system enhanced peroxidation of microsomal lipids. Vitamin E (alpha-tocopherol) inhibited lipid peroxidation. These results indicate that singlet oxygen may mediate lipid peroxide formation in epidermal microsomes.

Induction of neonatal rat skin and liver aryl hydrocarbon hydroxylase by coal tar and its constituents.

Topical application of coal tar solution (USP) to neonatal rats resulted in the induction of skin and liver aryl hydrocarbon hydroxylase (AHH) activities. Furthermore indirect exposure of the animals to coal tar vapors resulted in induction of the enzyme in skin and liver. Cutaneous application of coal tar to pregnant rats resulted in induction of skin and liver AHH activity in both mothers and prenatal rats. Among several defined constituents of coal tar tested benzo(a)pyrene (BP), anthracene and acridine were found to have measurable induction effects on neonatal rat skin and liver AHH. These studies indicate that therapeutic coal tar solution as well as selected defined chemical constituents of coal tar are capable of altering the activity of AHH in skin and liver.