Dynamics of pigmentation induction by repeated ultraviolet exposures: dose, dose interval and ultraviolet spectrum dependence.

BACKGROUND: The dynamics of ultraviolet (UV)-induced melanogenesis have been well characterized for single UV exposures. However, our knowledge of the effects of repeated UV exposures on the development of new pigmentation is limited. OBJECTIVES: To characterize the dynamics and dose dependence of pigmentation induction by repeated UV exposures using two different UV sources. METHODS: A total of 40 healthy subjects participated in the study: 21 were exposed to a 5% UVB/95% UVA source and 19 were exposed to a 2% UVB/98% UVA source. Skin phototypes 2-3 were represented. Subjects were exposed one to three times per week. The minimal erythemal dose and minimal melanogenic dose of all subjects were determined, and both visual and instrumental observations of the development of pigmentation and erythema were recorded. RESULTS: Dark-brown pigmentation could be produced by a cumulative UV dose of 4200 J m(-2) given as 10 exposures over 5 weeks. However, comparable pigmentation could also be induced by a cumulative dose of 2900 J m(-2) given as eight exposures over 4 weeks. The lowest cumulative dose of 1900 J m(-2) given over 4 weeks produced moderate pigmentation. The 2% UVB source led to earlier and darker pigmentation than the 5% UVB source did for equally erythemogenic doses. CONCLUSIONS: These observations show that the dynamics of melanogenesis induced by repeated exposures depends on UV dose, dose interval and emission spectrum. They also indicate that increasing the UV dose above a certain level of cumulative exposure does not significantly increase the level of UV-induced pigmentation.

In vivo measurement of skin erythema and pigmentation: new means of implementation of diffuse reflectance spectroscopy with a commercial instrument.

BACKGROUND: Various physical, chemical and biological insults, including exposure to ultraviolet (UV) radiation, cause erythema and change in pigmentation in human skin. These reactions provide an important measure of the cutaneous response to the insult. OBJECTIVES: To present a new implementation of a method for objective in vivo measurement of erythema and pigmentation. METHODS: The method is based on acquisition of reflectance spectra in the visible range using a commercially available spectrophotometer. The probe of this instrument incorporates an integrating sphere that captures the light remitted from the skin in a wide range of angles. We corrected the acquired reflectance spectra for the contribution of specular reflections by an amount given by the Fresnel equation and verified this correction experimentally. This correction is particularly important when measurements are performed on heavily pigmented skin. The corrected reflectance spectra are then transformed into absorbance spectra. To analyse these spectra, we developed an algorithm which can be used to calculate apparent concentrations of oxyhaemoglobin, deoxyhaemoglobin and melanin. This method was tested in clinical studies of skin reactions induced by exposure to UV radiation. These experiments involved three groups of subjects with progressively darker complexion (constitutive pigmentation). Each group consisted of 10 subjects. Erythema was measured 1 day after UV exposure, and pigmentation (melanin content) 1 week later. Results Distinct apparent absorbance spectra were obtained for dark, intermediate and fair skin. There was good agreement between reconstructed spectra and experimental data at relevant wavelengths. Difference absorption spectra were able to show the dose dependence of UV-induced responses, and erythema and pigmentation values obtained by the spectroscopic method showed good correlation with those derived by subjective visual grading. CONCLUSIONS: The results demonstrate that the presented methodology provides an objective noninvasive way of measuring UV-induced reactions independently of the level of constitutive pigmentation.

HIV-positive patients differ from HIV-negative patients in indications for and type of UV therapy used.

BACKGROUND: Treatments using UV, UVB, or oral psoralen and UVA (PUVA) have been advocated for the care of HIV-infected persons with skin diseases. Concerns about the safety of these treatments exist. OBJECTIVE: We attempted to determine the characteristics of HIV infected persons receiving UV therapy and establish the reasons for and type of treatment administered. METHODS: During two 2-week periods, we prospectively ascertained basic information on all patients treated at 40 phototherapy clinics and detailed clinical information on patients known to be infected with HIV. RESULTS: We identified 3716 persons receiving UV therapy, including 311 known to be infected with HIV. When compared with patients not known to be infected with HIV, HIV-positive patients were significantly more likely to be treated with UVB rather than PUVA and were more likely to be treated for pruritic conditions rather than psoriasis. CONCLUSION: There were great variations in the relative reliance on UVB and PUVA among centers. There appears to be no agreement as to which type of UV therapy is optimal for patients infected with HIV. Most patients known to the treating clinician to be HIV positive are in the advanced stages of HIV disease. The number of persons with less advanced HIV disease receiving treatment remains unquantified but may be even more clinically important.

Ultraviolet therapy of HIV-infected individuals: a panel discussion.

Patients infected with the human immunodeficiency virus (HIV) frequently develop skin diseases that are responsive to ultraviolet (UV) radiation. Studies on the effects of UV on HIV and on the immune system in vitro and in transgenic animals have raised questions regarding the safety of UV exposure in these patients. In this article, invited experts address issues concerning the safety of ultraviolet therapy in HIV-infected patients by discussing their clinical and/or research experience.

Photosensitized decontamination of blood with the silicon phthalocyanine Pc 4: no activation of the human immunodeficiency virus promoter.

Photochemical decontamination of red blood cell concentrates (RBCC) with the silicon phthalocyanine Pc 4 and red light is being studied to enhance the viral safety of blood transfusion. Recent reports indicate that treatments with radiation and various phototsensitizing agents can activate the promoter of human immunodeficiency virus (HIV). This raises the possibility that an inadequate, sublethal photochemical treatment of RBCC could induce HIV in latently infected cells. This question has been addressed using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV promoter. In control studies, 8-methoxypsoralen (8-MOP) excited by UVA light caused activation of the HIV promoter in a dose- and time-dependent manner. At 0.1 microgram/mL of 8-MOP, maximal activation occurred with 18 J/cm2, 30 h after light exposure, With Pc 4 at 20 nM, over 90% of HeLa cells were killed after 24 h when exposed to 1 J/cm2 of red light. During that time interval and over a wide range of light doses no activation of the HIV promoter occurred. It is concluded that RBCC sterilization with Pc 4 and red light is unlikely to induce HIV production in latently infected cells.

Medical UV exposures and HIV activation.

This paper presents the first attempt to evaluate the potential of clinical UV exposures to induce the human immunodeficiency (HIV) promoter and, thus, to upregulate HIV growth in those skin cells that are directly affected by the exposure. Using the data for HIV promoter activation in vitro, we computed UVB and psoralen plus UVA (PUVA) doses that produce 50% of the maximal promoter activation (AD50). Then, using (a) literature data for UV transmittance in the human skin, (b) a composite action spectrum for HIV promoter and pyrimidine dimer induction by UVB and (c) an action spectrum for DNA synthesis inhibition by PUVA, we estimated the distribution of medical UVB and PUVA doses in the skin. This allowed us to estimate how deep into the skin the HIV-activating doses might penetrate in an initial and an advanced stage of UVB or PUVA therapy. Such analysis was done for normal type II skin and for single exposures. The results allow us to predict where in the skin the HIV promoter may be induced by selected small and large therapeutic UVB or PUVA doses. To accommodate changes in skin topography due to disease and UV therapy, our considerations would require further refinements. For UVB we found that, when the incident dose on the surface of the skin is 500 J/m2 (290-320 nm) (initial stage of the therapy), the dose producing 50% of the maximal HIV promoter activation (ADUVB50) is limited to the stratum corneum. However, with an incident dose of 5000 J/m2 (an advanced stage of the therapy), ADUVB50) may be delivered as far as the living cells of the epidermis and even to some parts of the upper dermis. For PUVA we found that, when the incident UVA doses are 25 or 100 kJ/m2 (320-400 nm) (an initial and an advanced stage of therapy, respectively), and the 8-methoxypsoralen concentration in the blood is 0.1 microgram/mL (the desired level), the combined doses to the mid epidermis (and some areas of the upper dermis) are well below the 50% HIV promoter-activating PUVA dose (ADPUVA50). Only under the worst scenario conditions, i.e. an exceptionally high drug concentration in the patient's tissues and localization of HIV in the nearest proximity to the skin surface, would the combined PUVA dose expected during photochemotherapy exceed ADPUVA50. These results suggest that the probability of HIV activation in the epidermis by direct mechanisms is higher for UVB than for PUVA treatment. However, complexities of the UV-inducible HIV activation and immunomodulatory phenomena are such that our results by themselves should not be taken as an indication that UVB therapy carries a higher risk than PUVA therapy when administered to HIV-infected patients.

Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-pol is down-regulated in the antisense cell line. Such a sensitization is anticipated only where beta-pol is rate-limiting in a DNA repair pathway. A number of agents were tested: cis-diamminedichloroplatinum II (cisplatin); 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU); ionizing radiation and the radio-mimetic drug bleomycin; the bifunctional alkylating agents nitrogen mustard and L-phenylalanine mustard (melphalan); the monofunctional alkylating agent methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. In the cases of cisplatin and UV radiation, a significant enhancement of cytotoxicity was observed. Damage as a result of both of these agents is thought to be repaired by the nucleotide excision repair (NER) pathway. The results suggest that, in this cell line, beta-pol is involved in and is rate-limiting in NER. We propose that down-regulation of beta-pol by antisense approaches might be used to enhance the cytotoxic effects of cisplatin and other DNA damaging chemotherapeutic agents.

Effects of psoralen plus UVA radiation (PUVA) on HIV-1 in human beings: a pilot study.

BACKGROUND: Laboratory data document the activation of the HIV-1 genome on exposure to UV radiation, including PUVA. The overall effects of UV radiation exposure on HIV-1 infection in human beings are unknown. OBJECTIVE: Our purpose was to observe CD4 cell counts and quantitative markers of HIV-1 load in late-stage HIV-1-infected human beings receiving PUVA for various cutaneous diseases. METHODS: Samples of peripheral blood were obtained on days 0, 14, 30, and 60 of PUVA administered in therapeutic doses. Number of CD4+ T lymphocytes was determined by flow cytometry, and HIV-1 load was measured by semiquantitative polymerase chain reaction for viral genome in peripheral blood mononuclear cells, semiquantitative RNA-polymerase chain reaction for HIV-1 RNA in serum, and determination of p24 in serum. RESULTS: No significant changes in the measurements were observed. CONCLUSION: This study did not detect a deleterious effect on CD4 cell count or HIV-1 load during 2 months of PUVA treatment for patients in late stages of infection, with low CD4 cell counts and high HIV-1 loads.

Reassessment of the differential effects of ultraviolet and ionizing radiation on HIV promoter: the use of cell survival as the basis for comparisons.

Effects of different radiation treatments on the human immunodeficiency virus-1 (HIV) promoter were reassessed for exposures comparable to those encountered in clinical or cosmetic practice, using survival of the host cell as a basis for comparisons. The exposures were performed with two ultraviolet radiation sources commonly used as medical or cosmetic devices (UVASUN 2000 and FS20 lamps), a germicidal (G15T8) lamp and an X-ray machine. The UVC component of the FS20 lamp was filtered out. The emission spectra of the lamps were determined. The characteristics of these sources allowed us to discriminate among effects of UVA1 (340-400 nm), UVB + UVA2 (280-340 nm) and UVC (254 nm) radiations. Effects of irradiation were ascertained using cultures of HeLa cells stably transfected with the HIV promoter linked to a reporter-chloramphenicol acetyl transferase-gene. The exposures used caused at least two logs of cell killing. In this cytotoxicity range, UVA1 or X radiations had no effect on the HIV promoter, whereas UVB + UVA2 or UVC radiations activated the HIV promoter in a fluence-dependent manner. Survivals following exposure to UVB + UVA2 or UVC radiation were (1) at the lowest measurable HIV promoter activation, 30 and 20%, respectively, (2) at one-half maximal activation, 6 and 3%, respectively and (3) at the maximal activation, 0.5 and 0.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the human immunodeficiency virus promoter by UVA radiation in combination with psoralens or angelicins.

The effects of mono- and bifunctional furocoumarins plus UVA radiation (PUVA and related treatments) on the human immunodeficiency virus-1 (HIV-1) promoter were studied using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV-1 promoter. The experiments were performed with three psoralens (5-methoxypsoralen, 5-MOP; 8-methoxypsoralen, 8-MOP; and 4'-aminomethyl-4,8,5'-trimethylpsoralen, AMT) and four angelicins (angelicin; 4,5'-dimethylangelicin, 4,5'-DMA; 6,4'-dimethylangelicin, 6,4'-DMA; and 4,6,4'-trimethylangelicin, TMA). The drugs alone and UVA radiation alone showed no effect on the HIV promoter. However, when the cells were incubated with the furocoumarins at 0.1-40 micrograms/mL and then irradiated, the HIV promoter was activated in distinct fluence ranges, i.e. (1) no promoter activity was discernible at low fluences (e.g. at 0.1 microgram/mL of 8-MOP up to 100 kJ/m2), (2) as the fluence was increased, the promoter activity increased to reach a maximum (10-50-fold with respect to the unexposed samples), and (3) as the fluence was further increased, the promoter activity decreased. Similar (although shifted on the fluence scale) patterns were observed with either > 340-nm UVA radiation or with UVA radiation contaminated with a small amount of UVB radiation (typical for PUVA lamps). The effective fluences were inversely related to the drug concentration. Experiments with 5-MOP and 8-MOP indicated reciprocity of the drug concentration and radiation fluence. The HIV promoter response patterns were similar for monofunctional angelicins and bifunctional psoralens.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectral effects in activation of the human immunodeficiency virus promoter by psoralens plus ultraviolet A treatment.

The effects of PUVA treatment on HIV promoter activation and cell killing in HIV cat/HeLa cells were studied using two UV sources, a UVASUN sunlamp and a UVAR Photoactivation Chamber. A 4 to 5 times higher dose of ultraviolet radiation was required from the UVASUN lamp than from the UVAR lamps: 1) to activate the HIV promoter in the presence of 0.1 or 1.0 microgram/ml 8-MOP and 2) to reduce cell survival to a level of 10%, in the presence of 0.1 or 1.0 microgram/ml 8-MOP. In addition, exposures performed with a fixed dose of 20 kJ/m2 at varying concentrations of 8-MOP, required a 4.7 times higher combined PUVA dose from the UVASUN lamp than from the UVAR lamps. Two possible sources of these differences were analyzed: (1) the presence of UVB + UVA2 (280-340 nm) in the radiation emitted by the UVAR, but not the UVASUN lamp, and its potential biological activity independent of 8-MOP, and (2) the difference in the overlap of the emission spectra of the two lamps with the absorption spectrum of 8-MOP. The area of overlap was higher for the UVAR lamp than for the UVASUN lamp by a factor of 4.6, which is close to the difference between these two lamps in induction of the HIV promoter and killing HeLa cells. This indicates that the effectiveness of a particular UVA source used in combination with 8-MOP can be predicted by its congruence to the absorption spectrum of the photosensitizing drug.

Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth.

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.

Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.

A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed.

Characterization of DNA polymerase beta mRNA: cell-cycle and growth response in cultured human cells.

DNA polymerase beta (beta-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. We used a cDNA probe to study abundance of beta-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the beta-polymerase transcript is "low abundance" and is neither cell-cycle nor growth phase responsive.

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.

Chromosomal location of the human gene for DNA polymerase beta.

Inhibition studies indicate that DNA polymerase beta has a synthetic role in DNA repair after exposure of mammalian cells to some types of DNA-damaging agents. The primary structure of the enzyme is highly conserved in vertebrates, and nearly full-length cDNAs for the enzyme were recently cloned from mammalian cDNA libraries. Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids, using portions of the cDNA as probe, indicates that the gene for human DNA polymerase beta is single copy and located on the short arm or proximal long arm of chromosome 8 (8pter-8q22). A restriction fragment length polymorphism (RFLP) was detected in normal individuals by using a probe from the 5' end of the cDNA, and this RFLP probably is due to an insertion or duplication of DNA in 20-25% of the population. This restriction site can be used as one marker for chromosome 8 genetic linkage studies and for family studies of traits potentially involving this DNA repair gene.