Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-α and intercellular adhesion molecule-1 expression

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.     

Induction of mutagenic DNA damage in human fibroblasts after exposure to artificial tanning lamps

Summary There is increasing concern about the adverse health effects associated with the use of sunbeds, particularly with respect to skin photocarcinogenesis. The induction of mutagenic DNA damage is a prerequisite for the development of skin tumours, and it is well established that direct types of damage such as cyclobutane pyrimidine dimers (CPDs) give rise to mutations in tumour suppressor genes and oncogenes. In addition, ultraviolet radiation may induce indirect types of DNA damage, including oxidative products, which are also potentially mutagenic. By using specific DNA repair enzymes (T4 endonuclease V and endonuclease III) and the comet assay we have been able to detect the induction of CPDs, oxidized or hydrated pyrimidine bases and single-strand breaks in cultured human fibroblasts (MRC-5) alter exposure for between 15s and 20 min on two different commercial sunbeds containing Philips ' Performance ' 100W-R or Philips TL80W/10R lamps. The ratio of endonuclease III to T4 endonuclease V sensitive sites varied substantially between the two lamps and was 3.3% and 18%, respectively. The sunbed containing the ' Performence ' 100W-R lamps was as potent at inducing CPDs as was natural sunlight in fine weather. These results establish that commercial tanning lamps produce the types of DNA damage associated with photocarcinogenesis in human cells, and complement epidemiological evidence Indicating the potential risk of using sunbeds.     

Survey of the variation in ultraviolet outputs from ultraviolet A sunbeds in Bradford

Concerns have been expressed for some time regarding the growth of the cosmetic suntanning industry and the potential harmful effects resulting from these exposures. Recently published work has appeared to confirm a link between sunbed use and skin cancer. A previous survey in Oxford some years ago demonstrated significant output variations, and we have attempted to extend and update that work. Ultraviolet A, UVB and blue-light output measurements were made on 50 sunbeds using a radiometer fitted with broad-band filters and detectors. A number of irradiance measurements were made on each sunbed within each waveband so that the uniformity of the output could also be assessed. UVA outputs varied by a factor of 3, with a mean of 13.5 mW/cm²; UVB outputs varied by a factor of 60, with a mean of 19.2 μW/cm²; and blue-light outputs varied by a factor of 2.5, with a mean of 2.5 mW/cm². Outputs fall on average to 80% of the central value at either end of the sunbed. Facial units in sunbeds ranged in output between 18 and 45 mW/cm². Output uniformity shows wide variation, with 16% of the sunbeds having an axial coefficient of variation > 10%. UVB output is highly tube-specific. Eyewear used in sunbeds should also protect against blue light.     

Effects of selenium compounds on induction of DNA damage by broadband ultraviolet radiation in human keratinocytes

Background Ultraviolet radiation (UVR), a ubiquitous environmental genotoxin for the skin, produces DNA damage. The trace element selenium induces synthesis of the glutathione peroxidase and thioredoxin reductase enzyme families. These selenoenzymes detoxify a range of toxic compounds generated by free radicals. Objectives To assess the effects of pretreatment of primary human keratinocytes with selenium on UVR-induced DNA damage. Methods Cells were irradiated with UVR from FS-20 lamps and were subjected to comet assay. Results Comet tail length due to UVR-induced T4 endonuclease V-sensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 35 +/- 4.5 microm (mean +/- SD) immediately after irradiation (time 0 h, 100%). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nmol L-1 selenomethionine or 50 nmol L-1 sodium selenite had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, selenite and selenomethionine protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-hydroxy-2-deoxyguanosine photoproduct formation. In this assay, irradiation of keratinocytes increased mean +/- SD glycosylase-specific comet tail length from 5 +/- 1.5 microm to 19 +/- 3.3 microm. Preincubation for 18 h with 50 nmol L-1 selenite abolished the UVR-induced increase in comet length. Preincubation with 200 nmol L-1 selenomethionine was similarly protective. Conclusions Selenite and selenomethionine protect keratinocytes from UVR-induced oxidative damage, but not from formation of UVR-induced excision repair sites.     

UVA1 genotoxicity is mediated not by oxidative damage but by cyclobutane pyrimidine dimers in normal mouse skin

UVA1 induces the formation of 8-hydroxy-2'-deoxyguanosines (8-OH-dGs) and cyclobutane pyrimidine dimers (CPDs) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA1 has not been clarified. We irradiated living mouse skin with 364-nm UVA1 laser light and analyzed the DNA damage formation and mutation induction in the epidermis and dermis. Although dose-dependent increases were observed for both 8-OH-dG and CPD, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C --> T transition at a dipyrimidine site. Moreover, these UV-specific mutations occurred preferentially at the 5'-TCG-3' sequence, suggesting that CpG methylation and photosensitization-mediated triplet energy transfer to thymine contribute to the CPD-mediated UVA1 genotoxicity. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA1 exposure. We also found differences in the responses to the UVA1 genotoxicity between the epidermis and the dermis: the mutation induction after UVA1 irradiation was suppressed in the dermis at all levels of irradiance examined, whereas it leveled off from a certain high irradiance in the epidermis.     

紫外线照射后生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用

目的：明确对UVA及UVB照射后皮肤成纤维细胞生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用。方法：紫外线照射人皮肤成纤维细胞,提取细胞上清液中的微囊泡,利用光散射分析技术鉴定分析微囊泡的大小及数量。将紫外线照射后生成的微囊泡与正常成纤维细胞共孵育,荧光酶标仪定量检测活性氧含量,流式细胞仪检测细胞凋亡率。结果：UVA及UVB照射后皮肤成纤维细胞释放的微囊泡数量及大小明显高于正常成纤维细胞释放的微囊泡。正常纤维细胞、UVA和UVB照射后的成纤维细胞与微囊泡共孵育后活性氧荧光值分别为（52.76±1.4347）、（82.60±4.082）和（85.94±6.264）,凋亡率分别为（3.260±1.732）%,（28.94±2.430）%和（34.48±2.718）%,细胞的氧化损伤和凋亡可被抗氧化剂逆转。结论：急性中长波紫外线照射可诱导皮肤成纤维细胞释放微囊泡进一步介导细胞的氧化损伤和凋亡。     

Individual photosensitivity of human skin and UVA-induced pyrimidine dimers in DNA

Delineation of the DNA-damaging properties of UVA radiation is a major issue in understanding solar carcinogenesis. Emphasis was placed in this study on the formation of cyclobutane pyrimidine dimers (CPDs), which are now well established as the most frequent UVA-induced DNA lesions in human skin. The yield of CPDs was determined by a chromatographic assay following ex vivo UVA and UVB irradiation of biopsies taken from either phototype II or IV volunteers. A clear correlation was found between the frequency of UVB-induced CPDs and both the phototype and the minimum erythemal dose (MED). Similar results were obtained for the induction of CPDs upon exposure to UVA. Moreover, an excellent correlation was observed for each donor between the yield of DNA damage induced by either UVB or UVA. These observations show that the key parameters driving UVA-induced formation of CPDs are attenuation of radiation in the skin and the number of photons reaching skin cells rather than the cellular content in photosensitizers. In addition, the results show that both MED and phototype are good predictors of the vulnerability of DNA toward UVB and UVA in the skin. This result is of importance for the identification of individuals to be extensively protected.     

Mechanisms of mutation formation with long-wave ultraviolet light (UVA)

Long-wave ultraviolet (UV) A light is able to damage DNA, to cause mutations, and to induce skin cancer, but the exact mechanisms of UVA-induced mutation formation remain a matter of debate. While pyrimidine dimers are well established to mediate mutation formation with shortwave UVB, other types of DNA damage, such as oxidative base damage, have long been thought to be the premutagenic lesions for UVA mutagenesis. However, pyrimidine dimers can also be generated by UVA, and there are several lines of evidence that these are the most important premutagenic lesions not only for UVB- but also for UVA-induced mutation formation. C-->T transition mutations, which are generated by pyrimidine dimers, are called UV-signature mutations. They cannot be interpreted to be solely UVB-induced, as they are induced by UVA as well. Furthermore, there is no consistent evidence for a separate UVA-signature mutation that is only generated with UVA. We hypothesize that a weaker anti-mutagenic cellular response, but not a different type of DNA damage, may be responsible for a higher mutation rate per DNA photoproduct with UVA, as compared with UVB.     

Sunbeds in current use in Scotland: a survey of their output and patterns of use

Spectral irradiances of 100 commercially available sunbeds in current use have been measured. Ultraviolet (UV) A and UVB doses from sunbed use have been calculated and compared with doses likely to be received from solar radiation. The majority of sunbeds use UVA fluorescent tubes for irradiating the body and filtered metal halide lamps, which have a higher proportion of UVA1, for the face. The average minimum erythemal dose per session is 0.80, but irradiances for particular models varied by a factor of two to three primarily because of decline in lamp output with age. The UVA dose from a session on a sunbed is similar to that which might be received from 20 to 30 min sunbathing at a Mediterranean resort or 1 h on a sunny day in Glasgow, while UVB doses are 20–25% of this level. Responses from 200 current users of the sunbeds indicate that 38% had skin types 1 and 2, that 17% had more than 100 annual sunbed sessions and that 35% rarely or never used the goggles provided.     

Response of psoriasis to sunbed treatment: comparison of conventional ultraviolet A lamps with new higher ultraviolet B-emitting lamps

BACKGROUND: Sunbeds fitted with conventional ultraviolet (UV) A lamps that have about 0.7% UVB emission are widely used by patients with psoriasis even though they are minimally effective. A new fluorescent sunbed lamp has been developed that emits a higher proportion of UVB (4.6%) than conventional lamps and also requires shorter exposure times to achieve equivalent erythema. OBJECTIVES: To perform a randomized, within-patient comparison of conventional sunbed lamps (Cleo Performance) with the new lamps (Cleo Natural) in the treatment of psoriasis. METHODS: A sunbed and canopy unit were modified to allow exposure to Cleo Performance lamps on one side of the body (front and back) and Cleo Natural lamps to the other side of the body. Two studies were done. In study 1, equal erythemal doses were given from the two lamp types. In study 2, equal exposure times were given. We treated 34 patients with psoriasis, giving 12 exposures over a period of 4 weeks. Assessment was made using a modified Psoriasis Area and Severity Index (PASI) score, individual plaque assessment and patient questionnaire. RESULTS: Fourteen patients completed each study. In study 1, there was no significant difference in median improvement in half-body PASI score for the two lamp types. In study 2, there was a significant difference in PASI score improvement between the two lamps (median Cleo Performance change minus median Cleo Natural change was - 2.20; 95% confidence interval - 3.75 to - 0.65; P = 0.006). CONCLUSIONS: That no difference in response was found when equal erythemal doses were given suggests that the spectral emission of the Cleo Natural lamp is of no greater advantage for clearance of psoriasis than conventional lamps. However, the Cleo Natural lamps are more erythemally powerful, and exposure times similar to those used in conventional sunbeds result in a significant improvement of psoriasis. The risk of non-melanoma skin cancer from different patterns of exposure to Cleo Natural lamps can be estimated using established numerical models.     

Influence of ultraviolet A on scheduled and unscheduled DNA synthesis by ultraviolet B.

After irradiating mouse epidermis in vivo with ultraviolet radiation (UV), autoradiography using 3H-thymidine was performed to study the effects of UV on scheduled and unscheduled DNA synthesis during a 7 day-period. Suppression of scheduled DNA synthesis by 100 mJ/cm2 of UVB was continued for about 24 h and induction of unscheduled DNA synthesis by 100 mJ/cm2 of UVB for about 6 h after irradiation. It was also confirmed that UVA induced DNA damage if a dose of UVA over 30 J/cm2 was used. When UVA was applied 1 h before UVB irradiation, a significant increase in sparsely labeled cells (SLC) was observed and the numbers of SLC increased in proportion to the total dosage of UV. So it was confirmed that UVA preirradiation enhanced DNA damage by UVB.     

The effect of whole-body sunbed ultraviolet A exposure on the pharmacokinetics of the photolabile drug nifedipine

The calcium antagonist nifedipine absorbs ultraviolet A (UVA) radiation and readily photodegrades in vitro to a toxic nitroso-pyridine photoproduct. We examined whether whole body exposure of normal subjects to sunbed UVA radiation would affect the pharmacokinetics of nifedipine. Eight healthy, male, Caucasian volunteers (phototypes I-III) participated in this ethically approved, randomised, cross-over study. Each subject attended on 2 occasions, one week apart, and on each occasion was given a single oral dose (10 mg) of nifedipine following which blood samples were collected at 0, 0.5, 1. 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 7 h. During one of the visits, 15 min after nifedipine ingestion, a whole-body UVA (sunbed comprising Philips R-UVA lamps) dose of 70% of the individual's predetermined minimal phototoxic dose was delivered over a period of 17-36 min. Plasma nifedipine levels were measured using a standard reverse-phase high-performance liquid chromatography method. The area under the plasma concentration-time curve (AUC) of nifedipine during the UVA irradiation session (median 206 ng x ml(-1) x h(-1)) was significantly higher than during the non-irradiation control session (median 174.5 ng x ml(-1) x h(-1)) (P=0.03; 95% C.I. for difference in medians 9.9 to 55.9 ng x ml(-1) x h(-1)). UVA irradiation did not significantly affect any of the other measured pharmacokinetic parameters (Cmax, t 1/2, tmax). We demonstrate that sunbed UVA irradiation does not lead to in vivo photodegradation of nifedipine in healthy humans after a single dose. The apparent increase in AUC during UVA irradiation may be due to slightly slower metabolism of nifedipine in the presence of toxic photoproduct(s) or due to blood distribution changes affecting liver blood flow.     

Variation in calibration of hand-held ultraviolet (UV) meters for psoralen plus UVA and narrow-band UVB phototherapy

BACKGROUND: Phototherapy units should regularly use hand-held ultraviolet (UV) meters to assess the output of treatment lamps, and these meters should be accurately calibrated. Several medical physics departments in the U.K. can calibrate UV meters traceable to national standards, but there is concern that there may be disagreement among departments. In particular, there may be difficulty in calibration for narrow-band UVB phototherapy lamps (TL-01). OBJECTIVES: To ascertain the level of agreement in UV meter calibration at expert centres in the U.K., and to survey methodology at these centres, consider sources of errors and to make recommendations on calibration methods. METHODS: The same UV meter with two detectors (for UVA and UVB) was calibrated by seven medical physics departments. A questionnaire on methods was also distributed and measured spectral outputs from each centre were examined. RESULTS: The calibration factors for the meter varied by +/- 18% for the UVA detector and by +/- 60% for the UVB detector (2 standard deviations). Six centres performed calibration using a spectroradiometer and one centre used a reference meter method. The spectra of lamps used for calibration were similar. For the spectroradiometric methods there were some differences in methodology and instrumentation that may account for the differences in calibration factors. CONCLUSIONS: UV meter calibration in the U.K. shows unacceptable variability, particularly for TL-01 lamps. An accuracy of around of +/- 10% would be clinically acceptable and should be technically achievable.     

UVA1 impairs the repair of UVB‐induced DNA damage in normal human melanocytes

The exact correlation between melanoma and sun‐light is still a controversially debated issue. Although natural sunlight contains various ratios of UVA and UVB, most investigators so far focused on the effects of single solar wavebands and neglected possible interactions. Therefore, in this study primary human melanocytes of three donors were simultaneously exposed to physiologic doses of UVA1 and UVB. Effects on apoptosis were analysed using annexin V assays and cell death ELISAs, and effects on DNA damage were investigated using southwestern slot blots. While UVA1 did not influence UVB‐induced apoptosis, UVA1 impaired the repair of UVB‐induced cyclobutane pyrimidine dimers (CPD) as the amount of CPD was 1.8 times higher in UVA1 + UVB than in UVB only exposed melanocytes six hours after irradiation. We conclude that UVA1 might contribute to melanomagenesis as it partially inhibits the repair of UVB‐induced CPD in human melanocytes while it does not affect UVB‐mediated apoptosis.     

Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin

UVA- and UVB-induced tans which were visually identical with each other were induced in separate sites on the lower back of 5 normal human volunteers of good tanning ability. Tanning was achieved by 4 exposures to UVA and UVB administered over an 8-day period. One week after the last exposure the protection afforded by the two types of tan against UVB-induced erythema and against UVB-induced DNA damage was measured. Protection against erythema was measured by comparison of the minimal erythema doses of UVB in tanned and untanned skin. Protection against DNA damage was assessed by comparing the numbers of endonuclease-sensitive sites in epidermal DNA extracted from biopsies taken from tanned and untanned sites exposed to the same dose of UVB. The UVB tans conferred significant protection (mean 2.98-fold) against UVB-induced erythema. UVA tans were not associated with significant protection (mean 1.4-fold). In contrast, both UVA- and UVB-induced tans were associated with a similar reduction in yield of endonuclease-sensitive sites in epidermal DNA (in UVA tan to 47% and in UVB tan to 45% of the yield in untanned skin). Protection conferred by the tans against erythema was therefore not paralleled by protection against DNA damage.     

A controlled study of ultraviolet A sunbed treatment of psoriasis

BACKGROUND: Ultraviolet (UV) A sunbeds are widely used by patients with psoriasis in an attempt to treat their skin disease. However, there is little evidence that UVA therapy improves psoriasis, and the long-term risks of sunbed exposure are not known. OBJECTIVES: To perform a randomized, placebo-controlled study of UVA sunbed therapy for psoriasis. METHODS: A sunbed and canopy unit was modified to allow UVA exposure on one side of the body (front and back), and 'placebo' visible light exposure on the other side of the body. We treated 38 patients with psoriasis, giving 12 exposures over a period of 4 weeks. Assessment was made using a modified Psoriasis Area and Severity Index (PASI) score, individual plaque assessment and patient questionnaire. RESULTS: In 17 patients (47%) the PASI score showed a greater reduction on the UVA side compared with placebo, in 11 patients (31%) no difference was recorded between the two sides, and in eight (22%) the improvement was greater on the placebo-treated side. Overall, the median pretreatment half-body modified PASI score was 4.4 units, reducing to 3.9 units on the UVA-treated side and 4.2 units on the placebo-treated side (P = 0. 044 for difference in response). Breakdown of the plaque score into the individual components of erythema, scale and thickness revealed significant improvement only with the score for erythema. Although the degree of improvement was small, 64% of patients felt that the response was sufficiently good that they would use a sunbed again to treat their psoriasis. CONCLUSIONS: Our results show that a short course of sunbed treatment does improve psoriasis in some patients, but that the degree of improvement is small.     

Photoadaptation to ultraviolet (UV) radiation in vivo: photoproducts in epidermal cells following UVB therapy for psoriasis

BACKGROUND: Ultraviolet (UV) radiation is mutagenic and induces specific DNA lesions in human skin that are often found at dipyrimidine sites. These photoproducts are likely to be biologically relevant regarding skin carcinogenesis, as p53 mutations in skin tumours are most often found at these UV radiation-specific sites within DNA. Psoriasis patients receiving long-term phototherapy are at an increased risk of non-melanoma skin cancers. OBJECTIVES: The aim of this study was to quantify DNA photoproducts in human epidermis in vivo following consecutive doses of UVB and to investigate variations in DNA damage according to skin type, UVB dose and age. METHODS: Eleven psoriasis patients receiving UVB phototherapy three times a week were recruited and underwent skin biopsies on a non-sun-exposed site before starting phototherapy and after three, nine and 18 UVB exposures. A biopsy was also taken at least 4 weeks after stopping phototherapy. DNA was extracted from separated epidermis and three types of photoproducts were quantified using a novel 32P high-performance liquid chromatographic technique. RESULTS: The mean level of cyclobutane dipyrimidine dimers (CPDs) after three doses of UVB (dose range 0.03-0.15 J cm-2) was 3.2 (range 0.8-8.9) photoproducts per 106 normal nucleotides for TT=T dimers and 4.5 (range 0-14) per 106 normal nucleotides for TT=C dimers. The mean levels of TT-C 6-4 photoproducts after three doses of UVB were very low (0.2, range 0-1.8). Overall, the levels of TT=T and TT=C reached a plateau at three exposures and were found to decrease for subsequent exposures despite increasing UVB doses. Skin type was negatively associated with mean levels of CPDs. However, significant differences in levels of photoproducts were seen between individuals, even after adjusting for skin type. No association was found between challenge dose of UVB and photoproduct yield in this study. CONCLUSIONS: This study showed a great individual variation in the accumulation of DNA photoproducts following exposure to repetitive doses of UVB. Photoadaptive responses of human skin involving DNA repair, tanning and epidermal thickening are likely to explain the overall lack of increase in DNA lesions throughout phototherapy. This in vivo study confirms that psoriasis patients produce a significant amount of DNA photolesions at suberythemal doses of UVB. Further work is needed to investigate which host factors are most likely to predict susceptibility to UV radiation-induced DNA damage.     

Baicalin protects human fibroblasts against ultraviolet B-induced cyclobutane pyrimidine dimers formation

Exposure to ultraviolet B (UVB) irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused DNA damage. Photocarcinogenesis is caused largely by mutations at the sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, a DNA damage model of UVB irradiation-induced fibroblasts was established. The immunocytochemical staining, immuno dot blotting and Western blotting were employed in the study. We demonstrated that pre-treatment of fibroblasts with Baicalin dose-dependently reduced the amount of UVB-generated CPDs. Compared with UVB irradiated cells, UVB-induced p53 accumulation was less pronounced in Baicalin-treated cells. Taken together, these results suggest that Baicalin prevent CPDs formation induced by UVB. Baicalin is therefore a promising protective substance against UVB radiation.