261
Bim'himu'~a et Biophy,~ica Acta. 1084 ( 19t~I ) 2fi I - 268 ~o 19Ol El,~;cv~crScience Publi..,hers B.V.i.)005-27~t)/91/S03.5~) ,4DONi.~ (M}0527fi0t}I~lt213N
BBALIP 53683
UVA-induced lipid peroxidation in cultured human fibroblasts P a t r i c e Morli~re l, A n n i e M o y s a n ~, R e n 6 S a n t u s -', G a b r i e l e H i i p p e t, J e a n - C l a u d e Mazi~re _~.3a n d Louis D u b e r t r e t t l,aboratmrc de Dcrmatologie, hVSERM U. ,¢IZ th'~pitu/th'nri :~bmdor. Cn~teil (France), 2 Laboratoire de Ph~,'sico-Chim~e de l'A~htptation Bioh~gique, INSERM U. 31Z ),htst:ttt~l National d'Histoire Natl,.,~ellc, Paris (b'rimce~ and ¢ Laborute~ire de Bitschbnie. l:ac,lt~: de ,~h'dech~e Saint A~ltobt¢. Paris ( Fratu~')
(Received 9 October lt;t~lt)
Key words: UVA light: Endogenous photosen.~itizcr: Peroxidation: Membrane; Fibroblast
The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggest.s a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-a-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.
Introduction Numerous studies on the effects of sunlight on cells have shown the complexity of the involved photobio. chemical processes [1-5]. The main cellular target of UVB irradiation (280-320 nm) is DNA. The UVB-induced photodamage to DNA including cyclobutyl pyrimidine dimers, 6-4 photoproducts and strand breaks, trigger lethal and mutagenic effects and arc believed to be responsible for most skin cancers (for a review see Ref. 4). Although the long term effects of the UVA (320-400 rim) radiations on skin aging and skin cancer have been demonstrated [6,7], little is known about the molecular and cellular mechanisms responsible for these effects. Furthermore, the primary photochemical events triggering the short time responses such as erythema, inflammation and pigmentation arc poorly d o c u -
Abbreviations: BELT. butylaled hydroxytoluene: DFO. desterroxamine; EMEM, Earle's modified minimum essential medium: F(',~;, foelal calf serum; HBSS, Hanks buffered saline solution; L D | | . lactate dehydrogenase; SDS, sodium dodecyl .',ulfate; TBA. thiobarbituric acid: TBARS, thiobarblturic acid-reactive suhstances; TEP, tetraethoxypropane; Vit E, vitamin E (l~,t~-tt~copherol). Correspondence: P. Morli~:re, Laboratoirc dc Dermatologie. [NSERM U. 312, tl~pital tlenri Mondor. t~40IO Cr6teil. France.
mented. The involvement of free radicals is currently considered as most probable [8,9]. Both bacteria [10-12] and mammalian cells [13] can be inactivated by UVA exposure, An oxygen-dependence of these processes suggests that reactive oxygen species are involved in the lethal action of UVA on cultured human fibroblasts [14]. Moreover, some experiments suggest that free radicals are generated in skin by UVA [15,16]. On the other hand, the main DNA damage occurring during UVA exposure are single and double strand chain breaks probably resulting from radical species formation [4,5]. Although the induction of membrane damage by UVA has been sometimes linked to membrane lipid peroxidation, the only studies dealing with UVA-induced lipid peroxidation were performed, to our knowledge, on bacteria [17]. Related studies on cells wcrc carried out in presence of exogenous photosensitizers [18,19]. Membranes or membrane models with or without exogenous photosensitizers were also frequcntly considered [21)-22] (see also Ref. 23 for a review). The involvement of UVA-absorbing endogenous photosensitizers is obvious but their identity and the molecular events following the photosensitization arc still unknown [8,9,24]. To date it is frequently claimed, although not experimentally established, that lipid peroxidation occurs upon UVA irradiation of human cells in the absence of exogenous photosensitizer. This report provides evidencc that exposure of
262 cHltHred human fibroblasts to low dose Gf UVA, in the absence of any exogenous photosensitizer, triggers lipid pcroxidation. Experimental section
Chemicals, soh'ents, culture media and routbte equipDlC>tll
Hanks buffered saline solution (HBSS) was prepared without phenol red from the purest available chemicals (Merck, Sigma Chemical). Earle's modified minimum essential medium EMEM) without phenol red and containing 5 g/! Hepes was prepared from essential, non essential amino acids and vitamins obtained from Boehringer and from salts provided by Merck. Foetal caIf serum (FCS) was purchased from Boehringer, penicillin-streptomycin and trypsin from Flow Laboratory and amphotericin from Squibb. Thiobarbituric acid (TBA), l,l',3,Y-tetraethoxypropane (TEP), butylated hydroxytoluene (BHT), D,L-atocopherol (Vit El, pyruvic acid and reduced/3-nicotinamide adenine dinucleotide (NADH) were supplied by Sigma Chemical. Uvasol grade 1-butanol (butanol), ethanol, Hepes, Triton X-100, sodium dodecyl sulfate (SDS), trichloroacetic acid and ferric chloride were purchased from Merck and methanoI for HPLC from FSA Laboratory Supplies. Desferroxamine (DFO) was kindly provided by Ciba-Geigy. Water was obtained with a MilIipore Miili Q ion exchanger coupled to a Millipore Milli RO4 filtering unit. A Varian DMS 100 spectrophotometer and a Spex ] 12 spectrofluorometer were respectively used for absorption and fluorescence spcctroscopies. HPLC was run with a Varian Vista 5500 equipped with a Varian ultraviolet 200 detector, a Shimadzu RF 535 fluorescence HPLC monitor and a Varian 4290 integrator. Ce//cub,re Cultures of human skin fibroblasts obtained from breast plastic surgery were established in EMEM containing antibiotics (100 IU ml -~ penicillin, 100 /zg ml ~ streptomycin, 50 ng ml- ~ amphotericin) and supplcmcntcd with 10% FCS as earlier described [25]. Cells were further propagated in EMEM supplemcnted with 10% FCS without any other additive. For all experiments, cells in passages 4 to 8 were seeded at 60000 cells per 35 mm plastic Petri dishes (2.5 ml at 24000 cells/ml) and grown for about 7 days to reach conflucncy. Thus, each Petri dish contair.ed approx. 250000 cells corresponding to approx, t50/.tg protein.
Uttraciolct irradiation An ultraviolet-365 illuminating table (35 × 20 cm) equipped with 5 TF-20L tubes (Vilbert et Lourmat, France) was used for UVA irradiation. A glass window (4 mm thickness) placed 20 mm above the lamp re-
moved short wavelengths (transmittance < 0.0001 at 310 nm), Plastic petri dishes were placed or the glass window and were therefore irradiated from the bottom. The central area of the table allowed the homogeneous irradiation of 15 Petri dishes. Actinometry with potassium fen ioxalate gave an average light intensity of 3.8 mW cm - i.e., 7.0. l0 Is quanta s-J cm-- (min and max values are 3.5 and 4.1 mW cm -2 respectively).
Exposure of cells to uhrat'iolet light Cells were washed twice with 2 ml of HBSS, left with 1 mi of HBSS and irradiated with ultraviolet light. In parallel, other cells were sham-irradiated, i.e., kept in the dark during the same time and under the same environmental conditions as the irradiated cells. Then, the supernatant was removed and 900 /zl were kept frozen ( - 20°C) after addition of 90 #! BHT (2%, w / w in ethanol) for measuring thiobarbituric acid-reactive substances (TBARS) the following day (see below). When specified, cells were left in the dark for various times at 37°C after irradiation before supernatant collection. After supernatant collection, ceils were washed twice with HBBS and scrapped with a rubber policeman after addition of 500 lzi of water. Then 400/.tl of the disrupted cell solution were collected and added to 400/xl SDS (1%, w / w in water). 500/zl of the latter soWution were saved and kept fozen (-20°C) in the dark after addition of 50/~i I:iI-IT (2%, w / w in ethanol) for TBARS assay (see below). The protein content was measured [26] on the remaining 300/xl of SDS solubilized cell solution. In some experiments the effect of UVA on the LDH release was estimated in parallel to TBARS measurements. In this case the supernatant was collected for LDH analysis and ceils were scrapped after addition of 500 p.l of water. Then 500 #1 of triton X-100 (1%, w / w in water) were added and 100/zi of this mixture were diluted to ! ml with water and saved for LDH analysis as described below. TBARS assay Thiobarbituric-reactive substances were assayed according to a slightly modified procedure earlier described [27]. The defrost supernatant and cell samples received, prior to TBA, 90 and 50 !~! of BHT, respectively. Then 1 ml of a 0.375% (w/w) TBA solution in 0.25 M HCI containing 15% (w/w) triehloroacetic acid was added. The mixture was heated at 8 0 ° C for 15 rain, cooled on ice and extracted with butanol. The organic phase was collected (or fluorescence analysis (A~xc = 515 nm, A~m = 550 r,m). A TEP sample quantitatively yielding the m~londialdehyde-thiobarbituric acid (MDA-TBA) adduc~ was used for calibration of fluorescence data. Data are expressed in term of MDA equivalents normalized to the cell protein content. Unless otherwise specified the above conditions are the standard exper;mental conditions. The TBA assay
263 was also performed without BHT or in the presence of 150/zM DFO. The T E P cahbration was not modified by both conditions. Each data is the average _+ S.D. of triplicate measurements, each performed with one Petri dish.
HPLE mal.vsis of TBARS In some experiments, the T B A R S were assayed by both fluorescence and HPLC. In the later case, the reaction mixture was not extracted with butanol (:~ee above) but neutralized with 1 M NaOH (approx. 1.5 ml) and centrifuged for 5 rain at 10000 rpm before HPLC anaiysis. The MDA-TBA adduct from the T E P sample treated under the same conditions was used as standard. The HPLC was performed according to a slightly modified procedure previously described [28]. The samples (100 t-tl) injected in a/.t-Bondapak (W:ztcr,~ Associates) Cns reversed-phase column (3,9 m m × 30 cm) equipped with a guard-PAK column were elutcd with a mixture of methanol and 50 mM (pH 6.8) phosphate buffer (48:52, v / v ) at a flow rate of (L5 m l / m i n . Excitation and emission wavelengths of the fluorescence detector were set at 515 and 550 nm, respectively. Each data is the average + S . D of triplicate measurements, each performed with one Petri dish.
LDH analysis The LDH content of samples (supernatant or "cell" solution) was spectrophotometrically estimated by following the disappearance of N A D H during the LDHcatalyzed conversion of pyruvate to lactate [29]. This assay was performed immediately alter sample collection. To 2.05 ml of sodium pyruvate (0.3 mM in 50 mM (pH 7.5) phosphate buffer) were added 50/11 of NADH (8 mM in 50 mM (pH 7.5) phosphate buffer) and 900 ,ul of the supernatant or diluted cell solution. The loss of absorbance at 366 nm with time was measured. The LDH release was expressed as the percentage of LDH in the supernatant versus total LDH (LDIt in the supernatant + LDH in the "cell" solution). Each data is the average + S.D. of triplicate meas, rcmcnts, each performed with one Petri dish.
Results As illustrated in Fig. I, T B A R S arc significantly produced when cultured human fibroblasts arc exposed to UVA, Interestingly, T B A R S were detected not only in the cells (Fig. la) but also and to a much higher extent in the supernatant (fig. lb) which indicates that T B A R S are released from the cells during the irradiation. This release from irradiated cells is much higher than that from sham-irradiated cells. All unirradiated cell samples displayed low T B A R S Icvels but no significant difference (Fig. la) was detected
0.6
-~ 0.5 i
O~a 0.3
i
02
il
0.0
U! ! 0.5 .! 1,0 I 1,5 12,0 ! 2.5 ! 3.0 SI 3.0 4.0
®
"~ 3,0 ~
2,0
i
1.0 0.0 10.5 !1.0 !1.5 12.0 12.5 13.0 S13.0
Fig. I. hltracctlular (a) and exlracellular (hi TBARS produced upon UVA irr;tdkttitm of cultured human fibroblasls. TBARS in cells (a) and in the ~,upcrnatantIb) ',,,~¢r¢assayed immediatelybefore irradialion (UI. control |or cell samples only), after t/2 to 3 h irradiations {I 0.5 to i 3.0. rcspectb;ely)or after a 3 h sham irraddiation ISI 3.t)). TBARS arc expressed in MDA cquivalenl normalized Io the cell protein content, Fluorite rate ,vus 3.8 rrlW cm 2 Dala are mean_+ S.D, ol triplicates, See text for further experimenlM details.
between sham-irradiated cells (up to 3 h) and control cells (celts assayed without sham-irradiation period). Thus, a 3 h d~trk period in HBSS does not contribute to T B A R S production. In parallel to fluorometric determination, TBA assays on samples (supernatant cr cells) collected from irradiated (I h) or unirradiated cells (sham irrauiated) were carricd out by HPLC before butanol extraction (sec experimental section). The HPLC clution profiles of samples exhibit a single peak whose retention time (8.9 rain) is the same as the reference MDA-TBA adduct peak 'unpublished data). Fluorescence emission (A ...... = 55{) rim) and excitation spectra (A .... = 530 nm) of samples obtained from irradiated cells also agree fairly well with those ~f the reference MDA-TBA adduct (unpublished data). Moreover, quantitative analysis of HPLC and fluorometry data are in good agreement, cspecially for supernatant samples (see Table I). In the cell samples, fluorometric determinations are slightly overestimated with respect to HPLC measurements (see Table II). This suggests that substances other than MDA or those icading to MDA during T B A assay are fluorometricaHy measured. How-
264 TABLE I
3O
Quantitatire fluorometric aml HPLC determinations v f extracelhdar avid imracelhdar TBARS production tq;ott UJ:A ir.adiation of ctdtrtred
T
tnmran fibrohlasts
Cells were irradb, ted fi~r I h (13.7 J cm 2t and samples (supernatant and cells) were assayed after TBA reaction either by fluoromcl~' ~,fter butanol extraction or by HPLC before butanol extraction. See lext for details. TBARS expressed in MDA . quivalent normalized to tht; cell protein content (pmole/gg protein) ,are mean+S.D, of lripliculcs. Sham irradiated samples Irradiated samples supcr~ n:a:mt
cells
supernatant
0 !0.5 ll.O ll.5 12,0
cells
Fluorescence
data I-[PLC data
0 . 0 2 + _ 0 . 0 1 (}.17+(}.03
I,I)1 ±(}.l.lO 0,41 _[).02
I).(}l ± O.Ol
0.95 + 0.{}7
(l,[)9 ± 0.02
0.31 ± 0.03
evcr, as shown by the difference between fluorometric and HPLC determinations, these subgtances are in small and constant amounts in irradiated and unlrradlated samples as well, Irradiations were performed in the presence of glucose to minimize cell ~suffering'. They were also conducted in HBSS without glucose. Both conditions led to the same TBARS concentrations in samples from sham-irradiated or irradiated celIs (unpublished data). The TBA assay was routinely performed in the presence of BHT to impede further peroxidation of residu:~l unsaturated lipid during the assay. This assay was also carried out in the absence of B H T a n d in s o m e e x p c r i m e n t s in t h e p r e s e n c e o f d e s f e r r o x a m i n e ( D F O ) , t h e well k n o w n f e r r i c ion c h e l a t o r w h i c h p r e v e n t s hydroperoxide decomposition d u r i n g a s s a y . T h e final B H T a n d D F O c o n c e n t r a t i o n s w c r c a p p r o x . 6.7 m M {approx. 0,15~, w/w) a n d 150 g M , respectively. W h e r e a s in s u p e r n a t a n t s n o s i g n i f i c a n t d i f f e r e n c e w a s o b s e r v e d with o r w i t h o u t B T H a n d / o r D F O , T B A R S
measured in cell samples without BHT were slightly greater than those measured with BHT. With D F O alone, T B A R S were identical to those without BHT. With both B H T and D F O , T B A R S levels were similar
to those obtained with BHT alone. As shown in Fig. lb, the appearance of TBARS in the supernatant is dose-dependent and mostly linear for doses up to approx. 40 J cm-2. in parallel, it can be seen in Fig. 2, that the T B A R S formation is accompanied by the release of L D H in the supernatant, suggesting that membrane damage occurred upon irradia-
tion. Interestingly, the LDH release is not proportional to the UVA exposure, it remains very low during the first hour of irradiation and, then, markedly increases at longer irradiation times. Table !il shows the values
'~ 3.0
i:.f]bct of BItT Qtppro.~'. O.15r;. w ~ w) und / or I)FO ",50 ~ M) m TBA tt.~:~tt)"on TBARS t~l#aslo'~'m('llt
('ells were irradiated for 1 h (13.7 J cm z) and samples (supernatant or cells) pooled prior to BItT and/or DFO addition were immcdialcly assayed for TBARS. TBARS are expressed in MDA equivalent n(~rmalizcd to the cell prolein content (pmole/~g protein). Super-
natant .r cell samNes from a few Petri dishes were respeclively pooled Ior the assay. Data are mean + S.D, of triplicate TBA detcrmimltion ~t" these po~flcd samples. Add i!ivcs i n T B A assay ,mnc
BIIT
I)FO
BI IT+_ DFO _
ExtraccHul',ff TBARS Intracellular "I'B~RS
I.~4 +.ft.(13 I.~9.(),118 I.~9+0,09 [1.44 +rl).ll'
[).37-:ViL{)l
1.93±(L08
{I.44 +11.02 ~.).37_+11.03
_
_
2,5 13.0 Sl 3,0
Fip. 2. Release of LDH in the supernatant of cultured human fibroblasts exposed to UVA irradiation. LDH release is equal to LDH in the supernatant versus total LDH (supernatant±cells). LDH within cells m,J in supernatants were assayed after 0,5 1o 3 h irradiations (l 0.5 to I 3.0. respectively) or after a 3 h sham irradiation (S1 0.5 and Sl 3.tD. Data were obtained with the same cell preparation as in Fig, t. Fluence rate was 3.8 mW cm -2. Data are mean + S.D. of triplicates.
...,,
TABLE II
!__
-i!ii:;i!iii:~ ~[]'=°'"'' Intracelhlar
es.
'~ 2.0
!
[0,0 ! 1.0 DP 1,0 DP 3.0
U!
I 1,0 DP !.0 DP 3.0
Fig. 3. tntracellular and cxtraccllular TBARS levels after incubation periods in the dark following a I h UVA irradiation of cuhured human fibroblasls. TBARS in cells and in the supernatanl were assayed bcfl~r¢ the ilradiation (UI, fl~r cell samples onlyk immediately after 'he I h irradiation (I 1.0) and after a i or 3 h dark period (DP 1.0, DP 3.()) following the irradiation. TBARS are expressed in MDA ..quivaIenl normalized to the cell protein content, Fluence was 13,7 J cm '. Data are mean+S.D, of triplicates.
205 T A B L E III
TflARS and LDH relea.w ht supernalant of cultured truman fibroblasts irradiated &rang ] or 2 h (3. 8 m IV cm ~ 2) for carious donors and sub-passage
TBARS are expressed in MDA equivalent normalized to the -ell protein content (pmole/lag protein) and LDH release expressed in percent of total LDH, Controls lkw L D t l release are I h ~ or 2 hZ sham irradiated cells. Data arc mean +_S,D, of triplicales, n.d., not determined, LDH release
Donor Donor Donor Donor Donor Donor Donor
t passage l passage l passage 1 passage 2 passage 3 passage 4 passage
5 5 6 6 5 7 5
* * ** **
TBARS
control
1h
2h
!h
2h
5.7+0,2 t .~.7+_ 1.0 = 8.9-1-0.2 t 6,0 + 2.11 " 2. l + 0.8 : 1.0_+ (1.3 -" 1,0 +_0.5 -"
7,1 ±0.2 7 q ± 1.6 42.7 _+4,0 11.7.4:. l.S 2,5 .+_11.4 1.3 := 0.2 1,9 + 0,6
n.d. n.d. n.d. 33.1) --2_2. ! 5+5 _+ 1.4 1.8 _+0.8 2t),() +_ l.~l
1.20±0.05 [),88_+ 0,12 3.85+_ 2.0 l . ~ _+0 2 0 0.62 _4-0.05 tl.68 __0.05 1.4t} + 0.20
n.d. n.d. n,d. 4.5O _+0.20
t.76 _+0.115 1.38_+0.10 2,84 __.0,21)
* ' * * Data o b t a i n e d with different experiment, e.g.. with different cell seedings.
of TBARS and LDH released in the supernatant after 1 h of irradiation for experiments carried out with ceils from various donors or cells from the same donor in differents passa~e~, The LDH release varic~ in a wider range than TBARS and always parallels the TBARS formation. Fig. 3 shows that TBARS continue to form after the irradiation. The TBARS release in the supernatant increased with post-irradiation incubations at 37°C, while TBARS levels in the cells decreased, but the total TBARS (supernatant + cells) increased with the incubation time following the ultraviolet exposure. Moreover, TBARS production in the supernatant and in the cells was smaller when cells were preineubated overnight (15 h) with O,L-a-tocopherol Wit E) in the culture medium (Fig. 4). This protective effect was
L® 1.0
1
®
,:g.
0.4 i
[,o.o
~
C
TI
Tt0 Sl
o.o
Idl
C
Tt
TIO Si
Fig. 4. Effect of Vii E on extracetlular4,a)or intraeellular~b) TBARS produced upon U V A irradiation of cultured h u m a n fibroblasts. T B A R S were d e t e r m i n e d in the s u p e r n a t a n t (al or cells (bl after a I
h irradiation of untreated cells (C) or cells preincubaled overnight (16 h) with 1 p M Vit E ( T I ) or 10 # M Vit E (Tlt~). U n i r r a d i a t e d (UI) or s h a m - i r r a d i a t e d cells (SI) (see text a n d Fig. I fl~r details)
were not treated with Vit E. Data for unlrradiated or sham irradiated cells treated with Vit E did not significantlydiffer from data obtained with corresponding untreated cells (unpublished dataL T B A R S are expressed in M D A equivalent normalized to the cell
protein content. Fluence was 13.7 1 cm -", Data are mean .+.S.D. of triplicates.
maximum (approx. q0.c~c)at Vit E concentration greater than l0 ttM. A protective effect of approx. 50% was already observed at 10 ~ M Vit E in the incubation mcdiun;,
Discussion it is known that not only the solar UVB but also UVA may induce photobioiogical responses such as inflammation, pigmentation, photoaging or photocarcinogenesis in human skin [1-6]. The primary mechanisms responsible for these responses are poorly understood and to date are believed to involve photosensitization and o ~ g e n free radical processes although it has never been clearly evidenced [8,9,30]. The consequences of lipid peroxidation may include structural and functional modifications of membranes characterized by altered fluidity, increased permeabi!!ty and inactivation of cellular enzymes and transporters (for reviews see Rof. 8, 31, 32). The consequences of oxyradicals formation can be more indirect, such as mutagenesis due to DNA damage [33,34]. As most free radical processes, lipid peroxidation involves three steps: initiation, propagation and termination phases (for a review see Ref. 31). The chemis:,y of fatty acid peroxidation is rather complicated [36-38]. Numerous breakdown products can be formed including malondia!deh~de (MDA). It is generated in a side reaction of propagation from L O P . . Pero~yl radicals convcrt to bicyclic endoperoxides which may furth,:r decompose into MDA and are therefore clyptic tc~rms of MDA [31,321. Our study shows that TBARS are produced when human cultured fibroblasts are exposed to UVA. The TBARS are not only detected in the irradiated cells. but also and to a larger extent, in the supernatant of irradiated cells. This TBARS formation suggests that UVA irradiation triggers lipid peroxidation in human fibroblasts. Because of its simplicity, the TBA assay
266 performed with photometric or fluorometric detections has bccn frequently used to assay MDA or c~yptie MDA and is thus considered as an index of peroxidation. However, it has been somewhat questioned because other substances may react under the assay conditions leading to pink-colored products exhibiting absorption properties similar to those of the MDA-TBA adduct, i.c., peak or shoulder towards 530 nm [39-41]. These products include alk-2-enals, atka-2,4-dienals, alkanats, fatty acid hydroperoxides or pyrimidines [3944J. Fatty acid hydroperoxides, 2- and 2,4-unsaturated aldehydes, involved in the peroxidation process were reported to moderately contribule to TBARS with respect to MDA [42-45]. The contribution of 2- and 2,4-unsaturated aldehydes and also of various saturated aldehydes to MDA-TBA adduct was shown to be enhanced by the presence of sucrose, fructose and to a lesser extent of gtucose [39]. Since no significant differencc in TBARS was observed with or without glucose in HBSS, these data suggest that unsaturated aldehydes poorly contribute to TBARS formation. In the present study, the excitation and emission spectra of TBARS, identical to those of the reference MDA-TBA adduct, strongly suggest that they are actuai MDA-TBA adducts. Identification of the TBARS as MDA or reactive substances leading to MDA under TBA assay conditions (acidic medium, high temperaturc) was confirmed by HPLC analysis [28]. From the HPLC retention times it may be concluded that the MDA-TBA adduct is effectively assayed in the supernatant or cells. Since, in the supernatant, fluorometric rcsult:~ and HPLC determinations are in good agreemcnt, we ma:., conclude that the fluorometric determination is rcliabJe. In cell samples, low levels of TBARS othcr than those leading to the MDA-TBA adduct are fluorometically detected in irradiated and unirradiated cclls, but the irradiation does not enhance these non MDA-TBA adducts. No major difference was observed in the supernatant when BHT was omitted from the TBA test. It can bc concluded that thc pcroxidation of free fatty acid dt~ring ~he TBA assay does not significantly contributc to the mcasured TBARS in the supernatant, suggesting very low level of LH or of contaminating fcrric ions. The LOOH themselves could contribute to MDA and TBARS formation during the TBA test in the presence of contaminating fcrric ion.'~, although this contribution has been reported to be small with respect to cquivalcnt amounts of purc MDA [43-45]. No significant decrease of TBARS levels in the supernatant was observed when the TBA assay was conducted in the prescncc of DFO. Dcsferroxaminc, as avcry strong fcrric ion chclator, would inhibit LOOH decomposition. It may thus be concluded that, in these samples, MDA or cryptic MDA (endoperoxidcs) are effectively assayed, intcrcstingly, if the cndopcroxidic form of
MDA was assayed in the supernatant, it would mean that phospholipases must have split the peroxidized lipid to release the fatty acid moiety. However, in the absence of determinant data, this remains speculative. In cell samples, the absence of BHT in the TBA assay slightly increased TBARS levels, whereas the addition of DFO slightIy decreased them which again points to the low level of contaminating ferric ions and to the role of MDA or cryptic MDA in TBA assay. It should be noted the measured levels may be underestimated since MDA could be lost because it is volatile or metabolized or because it reacts with various substrates in the cells particularly with NH., groups such as aminophospholipids, amino acids, proteins or nucleic acids [46,47]. However, it is worth noting that part of MDA reacting with NH 2 groups to form imminopropene is recovered during the TBA assay [39,40]. As illustrated in Fig. 2, low UVA exposures (lower than about 15 J / c m 2) induced weak membrane damage as suggested by the low LDH release in the supernatant. Higher UVA doses result in higher MDA release in the supernatant and MDA levels in the cells, and in parallel to a much higher LDH release. Consequently, there exists threshold values for TBARS (approx. 0.3 and approx. 1.7 pmole/p,g cell protein, produced in the ceils and released in the supernatant, respectively) above which a significant LDH release is associated. The production of MDA and the membrane damage shown by the LDH release already support the view of a UVA-induced lipid peroxidation. This is further supported by the increase in MDA during dark periods subsequent to the UVA exposure (see Fig. 3). Indeed, once initiated lipid peroxidation is, expected to continue when the triggering events stop [48]. Another argument in favor of a UVA-induced lipid peroxidation is the inhibitory effect of Vit E, the well-known membrane-localized antioxidant [49], on MDA production. Vitamin E is a chain-breaking antioxidant inhibiting the propagation chains by reducing LOO. radicals. The inhibition of UVA-induced lipid peroxidation by Vit E is already observed at a concentration of 1 p,M in the incubation medium. Hanson and DeLeo have reported that UVA radiation stimulates araehidonic acid release and cyclooxygen~se activity in mammalian cells in culture [50]. Thus, MDA or endoperoxidic forms of MDA such as prostaglandin G2 can be generated as a result of the activation of phospholipase A2 and of the cyclooxygcnase pathways both involved in the biosynthesis of eicosanoids. They may therefore contribute to TBARS production and also to the LDH release reported above. However, it may be anticipated that this contribution might be small because of the strong inhibition of TBARS production by the chain-breaking antioxidant Vit E. But in the absence of definite evidence this vicw remains speculative because Vit E is sometimes
267 considered as a moderate inhibitor of cyclooxygenase [511. Interestingly, MDA production for ,he same UVA dose (13.7 J/cm-') varies in a rather large range for irradiated cells originating from differents donors or from the same donors but in different passages (see Table II11. The associated LDH relcase fairly well parallels the MDA production (see Table I!I) and significant LDH release are always associated with MDA levels which are higher than the above mentionned threshold. Therefore, data in Table !II suggest that, depending on cell condition or origin, this threshold is reached for different irradiation times, Although we have no data to support any definite conclusions, it may be speculated that such a behavior is related to the cellular defenee mechanisms against lipid peroxidation. In view of these results, the defence mechanisms may vary from one donor to another, which is rather likely, but also for a given donor within various passages, Since culture conditions were fairly'well standardized, performed in the absence of any additives (antibiotics, antifungi, ,~henol red) and experiments carried out with confluent cells in early passages (4 to 10) with a constant delay between cells sccding and irradiation, we are forced to admit that the cell status is a crucial parameter with respect to the U V A oxidative stress.
Concluding remarks The present experiments demonstrate that U V A irradiation of cultured fibroblasts triggers a lipid peroxidation process. Whether this peroxidation is a direct consequence of U V A exposure or the result of the activation of the prostaglandin cascade is under investigation although the strong inhibition of lipid peroxidation by Vit E tends to support the direct pathway. To our knowledge, this is the first report demonstrating UVA-induced lipid peroxidation in human cultured skin cells in the absence of any exogenous photosensitizer, it obviously requires endogenous chromophores such as riboflavins, N A D P - N A D P H , kynurenic acid, pterins, porphyrins; nevertheless, this critical target (s) has (have) not yet been identified (see Ref. 9 for a recent review). The secondary photochemical events leading to the induction phase of lipid peroxidation are even more unknown. Recent studies indicate that singlet oxygen, more than hydroxyl radical or superoxide anion, play an important role in the inactivation of human fibroblasts by U V A light [14]. Singlet oxygen is able to induce lipid peroxidation process via its direct reaction with LH to form LOOH, as well as to cause DNA damage. Since M D A cannot be produced as a direct consequence of singlet oxygen reaction with LH, its production requires further propagation of the peroxidation process via possibly metal ion-catalysts. Ex-
perimcnts arc clearly needed to further clarify the identity of the involved activated species and the molecular paths of UVA-induced lipid l~eroxidation. Owing to the potential involvement of MDA in the formation of lipofuscins [46] and in reaction with DNA [47,52] further studies should be undertaken to confirm the production of M D A in the course of UVA-induced lipid peroxidation. Further attention should be paid to the formation of other peroxidized materials since it is generally considered that MDA is a minor by-product of lipid peroxidation in various peroxidizing systems [531.
References i ('oohil, T.P., Peak, M,J, and Peak, LG. (I'087) Phott~hem. Photobiol. 46, 1043- i',1511. 2 Gangc, R,W. and Rosen, C.F. (!'080) Pholochcra. Pholobiol. 43, 7111-7115, 3 Kantor, G.J. (It}85) Photochem. Photobiol, 41,741-7-16. 4 Moan, I. and Peak, M.J.i.Iq891 J. Pholochem. Photobiol. B: Biol. 4, 21-34_ 5 Peak, M.J. und Peak, J,G, (1080,1 in The Biological Effects of UVA Radialiop (Urbach. F. and Gange, R.W., cds), pp. 42-56. Pracger, Nexx York. 6 Stabcrg, B.[,. and Wulf. |t.C. (lth',I3) J. lnves.:, Dermatol. 81, 5t7-5t0. 7 Van Vclden. H. and Van tier Leun, J.C. (1985; Pholochem. Photobiol. 42, 3119-4(13, S Black, FI.S. 119871 Photochem. Photebk~l 40,, 213-221, t~ TyrrelL R.M, and Keyse, S.M. (19901 J, Phottvchem. Phozobiol, B: Biol. 4, 34o-301. 1o Mo5.~, S,1-1. and Smith, K.C. 11081) Pholochcm. Photobiol, 33, 2t13-210, II Tyrell, R.M. 11979) Acta Biol. Med. Germ. 38, 1!s9-t269. 12 Wcbb, R.B. (It)77)in Photochemistry and PhotobLomg: Reviews (Smilh, K.C., ¢d.), Vol. 2, pp. 16t~-262. Plenum Press, Ne'~ York. 13 Dant)ure, I t.J. and Tyrett, R.M. (1976)Pholochcm. Photobiol. ".~, 171-177. 14 Tyrcll, R.M. und Pidoux, M, 11089) Phott~'hem, Pholobiot. 4~), 407-412,
15 Melteft, t1., Diczel, W. and Sonnichsen, N. (t97h) Experentia 32, 1397-1308. If) Pathak, M.A. ,.nd StraUon, K. (1968) Arch. Biochem. Bit}phys. 123, 4~8-47~,, 17 Chamberlain, J, and Muss, S.H. 11987) Photochcm. Pholobiol. 45, 625-tGI1, IN Pathak, M.A, and Carraro, C. (19851 Photochem. Pholobiol. 4Is, 74. 19 Thomas, J.P. and Girotd, A.W. 11989) Cancer Rcs. 49, 1682-168h. 211 Mandal. T.K. and Chanerjec, S,N. (lqS0) Radial. Res. 83, 2911~
3O2. 21 Ding, A.tl. and Chan. P.C. 110841Lipids, It), 278-284. 22 Girotti, A.W., Thomas. J.P, and Jordan, J.E. (1985) Pholochcm. Photobioi. 41. 2t,7-276, 23 Gironi. A.W. (19~1) Photochem. Photobiof.51,497-509. 24 Tyrcll. R,M. and Pidoux. M. 119881 Pholochcm. Photohiol, 47, 4115-412. 25 Coulomb. B, Saiag, P., Bell, E., Beitburd, F., Lehreton, C., I ; ",lan, M. anti Dubertret, k. (1980)) Br, J. Dermatol. 114, 91 - I l l l .
2fl Lo~,"y, O.H.. Rosebrough. N.J. ;.rod Farr. A.L. 11951) J. Biol.
Chem. 193, 265-275. 27 Aust, S.D. (11186)in Handbook of Methods for Oxygen Radical
268
28
29
30 31 32 33 34 35 36 37 38 39 4f)
Re~,earch (Grennwa!d, R.A., ed), pp. 203-207, CRC Press, Boca Raton. Wong, S.H.Y.. Knight, J.A., Hopfer, S.M.. Zaharia, O.. Leach. C,N.. Jr. and Sunderman, F,W.. Jr. (19871 Clin. Chem. 33, 21422(|. Bergmeyer. H,U, Bernt, E. and Hess. B. tl9b3) in Methods of Enzymatic Analysis (Bermeyer, H,U.. ed). pp. 736-741, Verlag Chemic. Berlin, Black, f-I.S,. Lenger, W,A,. Gerguis. J. and Thornby, J.i. (1985) Cancer Res. 45. 6254-6259. Girotti, A.W. ( 19851 J, Free Rad. Biol. Med. 1, 87-95, Pryor W.A, 11978) Photochem. Photobiol. 28. 787-801. Brown. K. and Fridovich, i. (198I) Arch. Biochem. Biophys. 2(16, 414-4t9, Suunmerfield, F.W. and Tappel, A.L. (19841 Anal. Biochem, 143. 265-27 I. Minotti, G. and Ausl. S.D. (1981) Chem. Phys. I,ipids 44, 191-2(]8. Frankel. E.N, 1,19851 Prog. Lip. Re'.;. 23, 197-22l. Tappel E.N. (19801 in Free Radical in Biology (Pryor, W.A., ed.), Vol. 4. pp. 1-45, Academic Press. New York. Kappus, tt. (19851 in Oxidative Stress (Sies, E., ed). pp. 273-31(I, Academic Press, Orlando, Knight. J.A., Pieper, R.K. and Mc Clellan, L. (19881Ciin. Chem. 34, 2433-2438. Bird, R.P. and Draper, H.H. (19841 Methods Enz3'mol. 105, 299-305.
41 Draper, H.H. and Hadley, M, (199(I) Methods Fnzymol. 186. 421-43 I. 42 Bigwood. T. and Read, O. (1989) Free Rad. Res. Comms. 6. 387-39_.2.
43 Kosugi. H. and Kikugawa, K. (1989) Free Rad. Biol. Med. 7. 205-2(17.
44 Outteridge, J.M.C. and Quinlan, G,J. (19831 J. Applied. Biochem. 5. 293-299. 45 Esterhauer, H. and Cheesman. K. (199111 Methods Enzymol. 186, 4t17-42 I. 46 Tsushida, M., Miura, T, and Airaba, K. (19871 Lipids 4, 297-325. 47 Vaca, C,E.. Wilhelm. J. and Harms-Ringdahl. M. (19881 Mut, Res. t 95, 137-149. 48 Reyftman, J-P,, Kohen. E., Morli~re, P., Santus, R., Kohen, C., Mangel. W.F., Dubertret. L. and Hirscb.berg. J.G. (19861 Photoehem. Photobiol. 461-469. 49 Niki. E. (19871 Chem. Phys. Lipids. 44, 227-253. 50 Hanson, D.L and DeLed, V.A. (19891 Photochem. Photobiol. 49, 423-430. 51 All, M.. Gudbranson. C.O. and McDonald. J,W.D. (19801 Proslagtandins and Medicine 4. 79-85. 52 Mukai, F.H. and Goldstein, B.D. (1976) Science 191,868-869. 53 Halliwelt, B. and Guttridge, J.M.C. (19901 Methods Enzymol. 186, 1-85.
Bim'himu'~a et Biophy,~ica Acta. 1084 ( 19t~I ) 2fi I - 268 ~o 19Ol El,~;cv~crScience Publi..,hers B.V.i.)005-27~t)/91/S03.5~) ,4DONi.~ (M}0527fi0t}I~lt213N
BBALIP 53683
UVA-induced lipid peroxidation in cultured human fibroblasts P a t r i c e Morli~re l, A n n i e M o y s a n ~, R e n 6 S a n t u s -', G a b r i e l e H i i p p e t, J e a n - C l a u d e Mazi~re _~.3a n d Louis D u b e r t r e t t l,aboratmrc de Dcrmatologie, hVSERM U. ,¢IZ th'~pitu/th'nri :~bmdor. Cn~teil (France), 2 Laboratoire de Ph~,'sico-Chim~e de l'A~htptation Bioh~gique, INSERM U. 31Z ),htst:ttt~l National d'Histoire Natl,.,~ellc, Paris (b'rimce~ and ¢ Laborute~ire de Bitschbnie. l:ac,lt~: de ,~h'dech~e Saint A~ltobt¢. Paris ( Fratu~')
(Received 9 October lt;t~lt)
Key words: UVA light: Endogenous photosen.~itizcr: Peroxidation: Membrane; Fibroblast
The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggest.s a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-a-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.
Introduction Numerous studies on the effects of sunlight on cells have shown the complexity of the involved photobio. chemical processes [1-5]. The main cellular target of UVB irradiation (280-320 nm) is DNA. The UVB-induced photodamage to DNA including cyclobutyl pyrimidine dimers, 6-4 photoproducts and strand breaks, trigger lethal and mutagenic effects and arc believed to be responsible for most skin cancers (for a review see Ref. 4). Although the long term effects of the UVA (320-400 rim) radiations on skin aging and skin cancer have been demonstrated [6,7], little is known about the molecular and cellular mechanisms responsible for these effects. Furthermore, the primary photochemical events triggering the short time responses such as erythema, inflammation and pigmentation arc poorly d o c u -
Abbreviations: BELT. butylaled hydroxytoluene: DFO. desterroxamine; EMEM, Earle's modified minimum essential medium: F(',~;, foelal calf serum; HBSS, Hanks buffered saline solution; L D | | . lactate dehydrogenase; SDS, sodium dodecyl .',ulfate; TBA. thiobarbituric acid: TBARS, thiobarblturic acid-reactive suhstances; TEP, tetraethoxypropane; Vit E, vitamin E (l~,t~-tt~copherol). Correspondence: P. Morli~:re, Laboratoirc dc Dermatologie. [NSERM U. 312, tl~pital tlenri Mondor. t~40IO Cr6teil. France.
mented. The involvement of free radicals is currently considered as most probable [8,9]. Both bacteria [10-12] and mammalian cells [13] can be inactivated by UVA exposure, An oxygen-dependence of these processes suggests that reactive oxygen species are involved in the lethal action of UVA on cultured human fibroblasts [14]. Moreover, some experiments suggest that free radicals are generated in skin by UVA [15,16]. On the other hand, the main DNA damage occurring during UVA exposure are single and double strand chain breaks probably resulting from radical species formation [4,5]. Although the induction of membrane damage by UVA has been sometimes linked to membrane lipid peroxidation, the only studies dealing with UVA-induced lipid peroxidation were performed, to our knowledge, on bacteria [17]. Related studies on cells wcrc carried out in presence of exogenous photosensitizers [18,19]. Membranes or membrane models with or without exogenous photosensitizers were also frequcntly considered [21)-22] (see also Ref. 23 for a review). The involvement of UVA-absorbing endogenous photosensitizers is obvious but their identity and the molecular events following the photosensitization arc still unknown [8,9,24]. To date it is frequently claimed, although not experimentally established, that lipid peroxidation occurs upon UVA irradiation of human cells in the absence of exogenous photosensitizer. This report provides evidencc that exposure of
262 cHltHred human fibroblasts to low dose Gf UVA, in the absence of any exogenous photosensitizer, triggers lipid pcroxidation. Experimental section
Chemicals, soh'ents, culture media and routbte equipDlC>tll
Hanks buffered saline solution (HBSS) was prepared without phenol red from the purest available chemicals (Merck, Sigma Chemical). Earle's modified minimum essential medium EMEM) without phenol red and containing 5 g/! Hepes was prepared from essential, non essential amino acids and vitamins obtained from Boehringer and from salts provided by Merck. Foetal caIf serum (FCS) was purchased from Boehringer, penicillin-streptomycin and trypsin from Flow Laboratory and amphotericin from Squibb. Thiobarbituric acid (TBA), l,l',3,Y-tetraethoxypropane (TEP), butylated hydroxytoluene (BHT), D,L-atocopherol (Vit El, pyruvic acid and reduced/3-nicotinamide adenine dinucleotide (NADH) were supplied by Sigma Chemical. Uvasol grade 1-butanol (butanol), ethanol, Hepes, Triton X-100, sodium dodecyl sulfate (SDS), trichloroacetic acid and ferric chloride were purchased from Merck and methanoI for HPLC from FSA Laboratory Supplies. Desferroxamine (DFO) was kindly provided by Ciba-Geigy. Water was obtained with a MilIipore Miili Q ion exchanger coupled to a Millipore Milli RO4 filtering unit. A Varian DMS 100 spectrophotometer and a Spex ] 12 spectrofluorometer were respectively used for absorption and fluorescence spcctroscopies. HPLC was run with a Varian Vista 5500 equipped with a Varian ultraviolet 200 detector, a Shimadzu RF 535 fluorescence HPLC monitor and a Varian 4290 integrator. Ce//cub,re Cultures of human skin fibroblasts obtained from breast plastic surgery were established in EMEM containing antibiotics (100 IU ml -~ penicillin, 100 /zg ml ~ streptomycin, 50 ng ml- ~ amphotericin) and supplcmcntcd with 10% FCS as earlier described [25]. Cells were further propagated in EMEM supplemcnted with 10% FCS without any other additive. For all experiments, cells in passages 4 to 8 were seeded at 60000 cells per 35 mm plastic Petri dishes (2.5 ml at 24000 cells/ml) and grown for about 7 days to reach conflucncy. Thus, each Petri dish contair.ed approx. 250000 cells corresponding to approx, t50/.tg protein.
Uttraciolct irradiation An ultraviolet-365 illuminating table (35 × 20 cm) equipped with 5 TF-20L tubes (Vilbert et Lourmat, France) was used for UVA irradiation. A glass window (4 mm thickness) placed 20 mm above the lamp re-
moved short wavelengths (transmittance < 0.0001 at 310 nm), Plastic petri dishes were placed or the glass window and were therefore irradiated from the bottom. The central area of the table allowed the homogeneous irradiation of 15 Petri dishes. Actinometry with potassium fen ioxalate gave an average light intensity of 3.8 mW cm - i.e., 7.0. l0 Is quanta s-J cm-- (min and max values are 3.5 and 4.1 mW cm -2 respectively).
Exposure of cells to uhrat'iolet light Cells were washed twice with 2 ml of HBSS, left with 1 mi of HBSS and irradiated with ultraviolet light. In parallel, other cells were sham-irradiated, i.e., kept in the dark during the same time and under the same environmental conditions as the irradiated cells. Then, the supernatant was removed and 900 /zl were kept frozen ( - 20°C) after addition of 90 #! BHT (2%, w / w in ethanol) for measuring thiobarbituric acid-reactive substances (TBARS) the following day (see below). When specified, cells were left in the dark for various times at 37°C after irradiation before supernatant collection. After supernatant collection, ceils were washed twice with HBBS and scrapped with a rubber policeman after addition of 500 lzi of water. Then 400/.tl of the disrupted cell solution were collected and added to 400/xl SDS (1%, w / w in water). 500/zl of the latter soWution were saved and kept fozen (-20°C) in the dark after addition of 50/~i I:iI-IT (2%, w / w in ethanol) for TBARS assay (see below). The protein content was measured [26] on the remaining 300/xl of SDS solubilized cell solution. In some experiments the effect of UVA on the LDH release was estimated in parallel to TBARS measurements. In this case the supernatant was collected for LDH analysis and ceils were scrapped after addition of 500 p.l of water. Then 500 #1 of triton X-100 (1%, w / w in water) were added and 100/zi of this mixture were diluted to ! ml with water and saved for LDH analysis as described below. TBARS assay Thiobarbituric-reactive substances were assayed according to a slightly modified procedure earlier described [27]. The defrost supernatant and cell samples received, prior to TBA, 90 and 50 !~! of BHT, respectively. Then 1 ml of a 0.375% (w/w) TBA solution in 0.25 M HCI containing 15% (w/w) triehloroacetic acid was added. The mixture was heated at 8 0 ° C for 15 rain, cooled on ice and extracted with butanol. The organic phase was collected (or fluorescence analysis (A~xc = 515 nm, A~m = 550 r,m). A TEP sample quantitatively yielding the m~londialdehyde-thiobarbituric acid (MDA-TBA) adduc~ was used for calibration of fluorescence data. Data are expressed in term of MDA equivalents normalized to the cell protein content. Unless otherwise specified the above conditions are the standard exper;mental conditions. The TBA assay
263 was also performed without BHT or in the presence of 150/zM DFO. The T E P cahbration was not modified by both conditions. Each data is the average _+ S.D. of triplicate measurements, each performed with one Petri dish.
HPLE mal.vsis of TBARS In some experiments, the T B A R S were assayed by both fluorescence and HPLC. In the later case, the reaction mixture was not extracted with butanol (:~ee above) but neutralized with 1 M NaOH (approx. 1.5 ml) and centrifuged for 5 rain at 10000 rpm before HPLC anaiysis. The MDA-TBA adduct from the T E P sample treated under the same conditions was used as standard. The HPLC was performed according to a slightly modified procedure previously described [28]. The samples (100 t-tl) injected in a/.t-Bondapak (W:ztcr,~ Associates) Cns reversed-phase column (3,9 m m × 30 cm) equipped with a guard-PAK column were elutcd with a mixture of methanol and 50 mM (pH 6.8) phosphate buffer (48:52, v / v ) at a flow rate of (L5 m l / m i n . Excitation and emission wavelengths of the fluorescence detector were set at 515 and 550 nm, respectively. Each data is the average + S . D of triplicate measurements, each performed with one Petri dish.
LDH analysis The LDH content of samples (supernatant or "cell" solution) was spectrophotometrically estimated by following the disappearance of N A D H during the LDHcatalyzed conversion of pyruvate to lactate [29]. This assay was performed immediately alter sample collection. To 2.05 ml of sodium pyruvate (0.3 mM in 50 mM (pH 7.5) phosphate buffer) were added 50/11 of NADH (8 mM in 50 mM (pH 7.5) phosphate buffer) and 900 ,ul of the supernatant or diluted cell solution. The loss of absorbance at 366 nm with time was measured. The LDH release was expressed as the percentage of LDH in the supernatant versus total LDH (LDIt in the supernatant + LDH in the "cell" solution). Each data is the average + S.D. of triplicate meas, rcmcnts, each performed with one Petri dish.
Results As illustrated in Fig. I, T B A R S arc significantly produced when cultured human fibroblasts arc exposed to UVA, Interestingly, T B A R S were detected not only in the cells (Fig. la) but also and to a much higher extent in the supernatant (fig. lb) which indicates that T B A R S are released from the cells during the irradiation. This release from irradiated cells is much higher than that from sham-irradiated cells. All unirradiated cell samples displayed low T B A R S Icvels but no significant difference (Fig. la) was detected
0.6
-~ 0.5 i
O~a 0.3
i
02
il
0.0
U! ! 0.5 .! 1,0 I 1,5 12,0 ! 2.5 ! 3.0 SI 3.0 4.0
®
"~ 3,0 ~
2,0
i
1.0 0.0 10.5 !1.0 !1.5 12.0 12.5 13.0 S13.0
Fig. I. hltracctlular (a) and exlracellular (hi TBARS produced upon UVA irr;tdkttitm of cultured human fibroblasls. TBARS in cells (a) and in the ~,upcrnatantIb) ',,,~¢r¢assayed immediatelybefore irradialion (UI. control |or cell samples only), after t/2 to 3 h irradiations {I 0.5 to i 3.0. rcspectb;ely)or after a 3 h sham irraddiation ISI 3.t)). TBARS arc expressed in MDA cquivalenl normalized Io the cell protein content, Fluorite rate ,vus 3.8 rrlW cm 2 Dala are mean_+ S.D, ol triplicates, See text for further experimenlM details.
between sham-irradiated cells (up to 3 h) and control cells (celts assayed without sham-irradiation period). Thus, a 3 h d~trk period in HBSS does not contribute to T B A R S production. In parallel to fluorometric determination, TBA assays on samples (supernatant cr cells) collected from irradiated (I h) or unirradiated cells (sham irrauiated) were carricd out by HPLC before butanol extraction (sec experimental section). The HPLC clution profiles of samples exhibit a single peak whose retention time (8.9 rain) is the same as the reference MDA-TBA adduct peak 'unpublished data). Fluorescence emission (A ...... = 55{) rim) and excitation spectra (A .... = 530 nm) of samples obtained from irradiated cells also agree fairly well with those ~f the reference MDA-TBA adduct (unpublished data). Moreover, quantitative analysis of HPLC and fluorometry data are in good agreement, cspecially for supernatant samples (see Table I). In the cell samples, fluorometric determinations are slightly overestimated with respect to HPLC measurements (see Table II). This suggests that substances other than MDA or those icading to MDA during T B A assay are fluorometricaHy measured. How-
264 TABLE I
3O
Quantitatire fluorometric aml HPLC determinations v f extracelhdar avid imracelhdar TBARS production tq;ott UJ:A ir.adiation of ctdtrtred
T
tnmran fibrohlasts
Cells were irradb, ted fi~r I h (13.7 J cm 2t and samples (supernatant and cells) were assayed after TBA reaction either by fluoromcl~' ~,fter butanol extraction or by HPLC before butanol extraction. See lext for details. TBARS expressed in MDA . quivalent normalized to tht; cell protein content (pmole/gg protein) ,are mean+S.D, of lripliculcs. Sham irradiated samples Irradiated samples supcr~ n:a:mt
cells
supernatant
0 !0.5 ll.O ll.5 12,0
cells
Fluorescence
data I-[PLC data
0 . 0 2 + _ 0 . 0 1 (}.17+(}.03
I,I)1 ±(}.l.lO 0,41 _[).02
I).(}l ± O.Ol
0.95 + 0.{}7
(l,[)9 ± 0.02
0.31 ± 0.03
evcr, as shown by the difference between fluorometric and HPLC determinations, these subgtances are in small and constant amounts in irradiated and unlrradlated samples as well, Irradiations were performed in the presence of glucose to minimize cell ~suffering'. They were also conducted in HBSS without glucose. Both conditions led to the same TBARS concentrations in samples from sham-irradiated or irradiated celIs (unpublished data). The TBA assay was routinely performed in the presence of BHT to impede further peroxidation of residu:~l unsaturated lipid during the assay. This assay was also carried out in the absence of B H T a n d in s o m e e x p c r i m e n t s in t h e p r e s e n c e o f d e s f e r r o x a m i n e ( D F O ) , t h e well k n o w n f e r r i c ion c h e l a t o r w h i c h p r e v e n t s hydroperoxide decomposition d u r i n g a s s a y . T h e final B H T a n d D F O c o n c e n t r a t i o n s w c r c a p p r o x . 6.7 m M {approx. 0,15~, w/w) a n d 150 g M , respectively. W h e r e a s in s u p e r n a t a n t s n o s i g n i f i c a n t d i f f e r e n c e w a s o b s e r v e d with o r w i t h o u t B T H a n d / o r D F O , T B A R S
measured in cell samples without BHT were slightly greater than those measured with BHT. With D F O alone, T B A R S were identical to those without BHT. With both B H T and D F O , T B A R S levels were similar
to those obtained with BHT alone. As shown in Fig. lb, the appearance of TBARS in the supernatant is dose-dependent and mostly linear for doses up to approx. 40 J cm-2. in parallel, it can be seen in Fig. 2, that the T B A R S formation is accompanied by the release of L D H in the supernatant, suggesting that membrane damage occurred upon irradia-
tion. Interestingly, the LDH release is not proportional to the UVA exposure, it remains very low during the first hour of irradiation and, then, markedly increases at longer irradiation times. Table !il shows the values
'~ 3.0
i:.f]bct of BItT Qtppro.~'. O.15r;. w ~ w) und / or I)FO ",50 ~ M) m TBA tt.~:~tt)"on TBARS t~l#aslo'~'m('llt
('ells were irradiated for 1 h (13.7 J cm z) and samples (supernatant or cells) pooled prior to BItT and/or DFO addition were immcdialcly assayed for TBARS. TBARS are expressed in MDA equivalent n(~rmalizcd to the cell prolein content (pmole/~g protein). Super-
natant .r cell samNes from a few Petri dishes were respeclively pooled Ior the assay. Data are mean + S.D, of triplicate TBA detcrmimltion ~t" these po~flcd samples. Add i!ivcs i n T B A assay ,mnc
BIIT
I)FO
BI IT+_ DFO _
ExtraccHul',ff TBARS Intracellular "I'B~RS
I.~4 +.ft.(13 I.~9.(),118 I.~9+0,09 [1.44 +rl).ll'
[).37-:ViL{)l
1.93±(L08
{I.44 +11.02 ~.).37_+11.03
_
_
2,5 13.0 Sl 3,0
Fip. 2. Release of LDH in the supernatant of cultured human fibroblasts exposed to UVA irradiation. LDH release is equal to LDH in the supernatant versus total LDH (supernatant±cells). LDH within cells m,J in supernatants were assayed after 0,5 1o 3 h irradiations (l 0.5 to I 3.0. respectively) or after a 3 h sham irradiation (S1 0.5 and Sl 3.tD. Data were obtained with the same cell preparation as in Fig, t. Fluence rate was 3.8 mW cm -2. Data are mean + S.D. of triplicates.
...,,
TABLE II
!__
-i!ii:;i!iii:~ ~[]'=°'"'' Intracelhlar
es.
'~ 2.0
!
[0,0 ! 1.0 DP 1,0 DP 3.0
U!
I 1,0 DP !.0 DP 3.0
Fig. 3. tntracellular and cxtraccllular TBARS levels after incubation periods in the dark following a I h UVA irradiation of cuhured human fibroblasls. TBARS in cells and in the supernatanl were assayed bcfl~r¢ the ilradiation (UI, fl~r cell samples onlyk immediately after 'he I h irradiation (I 1.0) and after a i or 3 h dark period (DP 1.0, DP 3.()) following the irradiation. TBARS are expressed in MDA ..quivaIenl normalized to the cell protein content, Fluence was 13,7 J cm '. Data are mean+S.D, of triplicates.
205 T A B L E III
TflARS and LDH relea.w ht supernalant of cultured truman fibroblasts irradiated &rang ] or 2 h (3. 8 m IV cm ~ 2) for carious donors and sub-passage
TBARS are expressed in MDA equivalent normalized to the -ell protein content (pmole/lag protein) and LDH release expressed in percent of total LDH, Controls lkw L D t l release are I h ~ or 2 hZ sham irradiated cells. Data arc mean +_S,D, of triplicales, n.d., not determined, LDH release
Donor Donor Donor Donor Donor Donor Donor
t passage l passage l passage 1 passage 2 passage 3 passage 4 passage
5 5 6 6 5 7 5
* * ** **
TBARS
control
1h
2h
!h
2h
5.7+0,2 t .~.7+_ 1.0 = 8.9-1-0.2 t 6,0 + 2.11 " 2. l + 0.8 : 1.0_+ (1.3 -" 1,0 +_0.5 -"
7,1 ±0.2 7 q ± 1.6 42.7 _+4,0 11.7.4:. l.S 2,5 .+_11.4 1.3 := 0.2 1,9 + 0,6
n.d. n.d. n.d. 33.1) --2_2. ! 5+5 _+ 1.4 1.8 _+0.8 2t),() +_ l.~l
1.20±0.05 [),88_+ 0,12 3.85+_ 2.0 l . ~ _+0 2 0 0.62 _4-0.05 tl.68 __0.05 1.4t} + 0.20
n.d. n.d. n,d. 4.5O _+0.20
t.76 _+0.115 1.38_+0.10 2,84 __.0,21)
* ' * * Data o b t a i n e d with different experiment, e.g.. with different cell seedings.
of TBARS and LDH released in the supernatant after 1 h of irradiation for experiments carried out with ceils from various donors or cells from the same donor in differents passa~e~, The LDH release varic~ in a wider range than TBARS and always parallels the TBARS formation. Fig. 3 shows that TBARS continue to form after the irradiation. The TBARS release in the supernatant increased with post-irradiation incubations at 37°C, while TBARS levels in the cells decreased, but the total TBARS (supernatant + cells) increased with the incubation time following the ultraviolet exposure. Moreover, TBARS production in the supernatant and in the cells was smaller when cells were preineubated overnight (15 h) with O,L-a-tocopherol Wit E) in the culture medium (Fig. 4). This protective effect was
L® 1.0
1
®
,:g.
0.4 i
[,o.o
~
C
TI
Tt0 Sl
o.o
Idl
C
Tt
TIO Si
Fig. 4. Effect of Vii E on extracetlular4,a)or intraeellular~b) TBARS produced upon U V A irradiation of cultured h u m a n fibroblasts. T B A R S were d e t e r m i n e d in the s u p e r n a t a n t (al or cells (bl after a I
h irradiation of untreated cells (C) or cells preincubaled overnight (16 h) with 1 p M Vit E ( T I ) or 10 # M Vit E (Tlt~). U n i r r a d i a t e d (UI) or s h a m - i r r a d i a t e d cells (SI) (see text a n d Fig. I fl~r details)
were not treated with Vit E. Data for unlrradiated or sham irradiated cells treated with Vit E did not significantlydiffer from data obtained with corresponding untreated cells (unpublished dataL T B A R S are expressed in M D A equivalent normalized to the cell
protein content. Fluence was 13.7 1 cm -", Data are mean .+.S.D. of triplicates.
maximum (approx. q0.c~c)at Vit E concentration greater than l0 ttM. A protective effect of approx. 50% was already observed at 10 ~ M Vit E in the incubation mcdiun;,
Discussion it is known that not only the solar UVB but also UVA may induce photobioiogical responses such as inflammation, pigmentation, photoaging or photocarcinogenesis in human skin [1-6]. The primary mechanisms responsible for these responses are poorly understood and to date are believed to involve photosensitization and o ~ g e n free radical processes although it has never been clearly evidenced [8,9,30]. The consequences of lipid peroxidation may include structural and functional modifications of membranes characterized by altered fluidity, increased permeabi!!ty and inactivation of cellular enzymes and transporters (for reviews see Rof. 8, 31, 32). The consequences of oxyradicals formation can be more indirect, such as mutagenesis due to DNA damage [33,34]. As most free radical processes, lipid peroxidation involves three steps: initiation, propagation and termination phases (for a review see Ref. 31). The chemis:,y of fatty acid peroxidation is rather complicated [36-38]. Numerous breakdown products can be formed including malondia!deh~de (MDA). It is generated in a side reaction of propagation from L O P . . Pero~yl radicals convcrt to bicyclic endoperoxides which may furth,:r decompose into MDA and are therefore clyptic tc~rms of MDA [31,321. Our study shows that TBARS are produced when human cultured fibroblasts are exposed to UVA. The TBARS are not only detected in the irradiated cells. but also and to a larger extent, in the supernatant of irradiated cells. This TBARS formation suggests that UVA irradiation triggers lipid peroxidation in human fibroblasts. Because of its simplicity, the TBA assay
266 performed with photometric or fluorometric detections has bccn frequently used to assay MDA or c~yptie MDA and is thus considered as an index of peroxidation. However, it has been somewhat questioned because other substances may react under the assay conditions leading to pink-colored products exhibiting absorption properties similar to those of the MDA-TBA adduct, i.c., peak or shoulder towards 530 nm [39-41]. These products include alk-2-enals, atka-2,4-dienals, alkanats, fatty acid hydroperoxides or pyrimidines [3944J. Fatty acid hydroperoxides, 2- and 2,4-unsaturated aldehydes, involved in the peroxidation process were reported to moderately contribule to TBARS with respect to MDA [42-45]. The contribution of 2- and 2,4-unsaturated aldehydes and also of various saturated aldehydes to MDA-TBA adduct was shown to be enhanced by the presence of sucrose, fructose and to a lesser extent of gtucose [39]. Since no significant differencc in TBARS was observed with or without glucose in HBSS, these data suggest that unsaturated aldehydes poorly contribute to TBARS formation. In the present study, the excitation and emission spectra of TBARS, identical to those of the reference MDA-TBA adduct, strongly suggest that they are actuai MDA-TBA adducts. Identification of the TBARS as MDA or reactive substances leading to MDA under TBA assay conditions (acidic medium, high temperaturc) was confirmed by HPLC analysis [28]. From the HPLC retention times it may be concluded that the MDA-TBA adduct is effectively assayed in the supernatant or cells. Since, in the supernatant, fluorometric rcsult:~ and HPLC determinations are in good agreemcnt, we ma:., conclude that the fluorometric determination is rcliabJe. In cell samples, low levels of TBARS othcr than those leading to the MDA-TBA adduct are fluorometically detected in irradiated and unirradiated cclls, but the irradiation does not enhance these non MDA-TBA adducts. No major difference was observed in the supernatant when BHT was omitted from the TBA test. It can bc concluded that thc pcroxidation of free fatty acid dt~ring ~he TBA assay does not significantly contributc to the mcasured TBARS in the supernatant, suggesting very low level of LH or of contaminating fcrric ions. The LOOH themselves could contribute to MDA and TBARS formation during the TBA test in the presence of contaminating fcrric ion.'~, although this contribution has been reported to be small with respect to cquivalcnt amounts of purc MDA [43-45]. No significant decrease of TBARS levels in the supernatant was observed when the TBA assay was conducted in the prescncc of DFO. Dcsferroxaminc, as avcry strong fcrric ion chclator, would inhibit LOOH decomposition. It may thus be concluded that, in these samples, MDA or cryptic MDA (endoperoxidcs) are effectively assayed, intcrcstingly, if the cndopcroxidic form of
MDA was assayed in the supernatant, it would mean that phospholipases must have split the peroxidized lipid to release the fatty acid moiety. However, in the absence of determinant data, this remains speculative. In cell samples, the absence of BHT in the TBA assay slightly increased TBARS levels, whereas the addition of DFO slightIy decreased them which again points to the low level of contaminating ferric ions and to the role of MDA or cryptic MDA in TBA assay. It should be noted the measured levels may be underestimated since MDA could be lost because it is volatile or metabolized or because it reacts with various substrates in the cells particularly with NH., groups such as aminophospholipids, amino acids, proteins or nucleic acids [46,47]. However, it is worth noting that part of MDA reacting with NH 2 groups to form imminopropene is recovered during the TBA assay [39,40]. As illustrated in Fig. 2, low UVA exposures (lower than about 15 J / c m 2) induced weak membrane damage as suggested by the low LDH release in the supernatant. Higher UVA doses result in higher MDA release in the supernatant and MDA levels in the cells, and in parallel to a much higher LDH release. Consequently, there exists threshold values for TBARS (approx. 0.3 and approx. 1.7 pmole/p,g cell protein, produced in the ceils and released in the supernatant, respectively) above which a significant LDH release is associated. The production of MDA and the membrane damage shown by the LDH release already support the view of a UVA-induced lipid peroxidation. This is further supported by the increase in MDA during dark periods subsequent to the UVA exposure (see Fig. 3). Indeed, once initiated lipid peroxidation is, expected to continue when the triggering events stop [48]. Another argument in favor of a UVA-induced lipid peroxidation is the inhibitory effect of Vit E, the well-known membrane-localized antioxidant [49], on MDA production. Vitamin E is a chain-breaking antioxidant inhibiting the propagation chains by reducing LOO. radicals. The inhibition of UVA-induced lipid peroxidation by Vit E is already observed at a concentration of 1 p,M in the incubation medium. Hanson and DeLeo have reported that UVA radiation stimulates araehidonic acid release and cyclooxygen~se activity in mammalian cells in culture [50]. Thus, MDA or endoperoxidic forms of MDA such as prostaglandin G2 can be generated as a result of the activation of phospholipase A2 and of the cyclooxygcnase pathways both involved in the biosynthesis of eicosanoids. They may therefore contribute to TBARS production and also to the LDH release reported above. However, it may be anticipated that this contribution might be small because of the strong inhibition of TBARS production by the chain-breaking antioxidant Vit E. But in the absence of definite evidence this vicw remains speculative because Vit E is sometimes
267 considered as a moderate inhibitor of cyclooxygenase [511. Interestingly, MDA production for ,he same UVA dose (13.7 J/cm-') varies in a rather large range for irradiated cells originating from differents donors or from the same donors but in different passages (see Table II11. The associated LDH relcase fairly well parallels the MDA production (see Table I!I) and significant LDH release are always associated with MDA levels which are higher than the above mentionned threshold. Therefore, data in Table !II suggest that, depending on cell condition or origin, this threshold is reached for different irradiation times, Although we have no data to support any definite conclusions, it may be speculated that such a behavior is related to the cellular defenee mechanisms against lipid peroxidation. In view of these results, the defence mechanisms may vary from one donor to another, which is rather likely, but also for a given donor within various passages, Since culture conditions were fairly'well standardized, performed in the absence of any additives (antibiotics, antifungi, ,~henol red) and experiments carried out with confluent cells in early passages (4 to 10) with a constant delay between cells sccding and irradiation, we are forced to admit that the cell status is a crucial parameter with respect to the U V A oxidative stress.
Concluding remarks The present experiments demonstrate that U V A irradiation of cultured fibroblasts triggers a lipid peroxidation process. Whether this peroxidation is a direct consequence of U V A exposure or the result of the activation of the prostaglandin cascade is under investigation although the strong inhibition of lipid peroxidation by Vit E tends to support the direct pathway. To our knowledge, this is the first report demonstrating UVA-induced lipid peroxidation in human cultured skin cells in the absence of any exogenous photosensitizer, it obviously requires endogenous chromophores such as riboflavins, N A D P - N A D P H , kynurenic acid, pterins, porphyrins; nevertheless, this critical target (s) has (have) not yet been identified (see Ref. 9 for a recent review). The secondary photochemical events leading to the induction phase of lipid peroxidation are even more unknown. Recent studies indicate that singlet oxygen, more than hydroxyl radical or superoxide anion, play an important role in the inactivation of human fibroblasts by U V A light [14]. Singlet oxygen is able to induce lipid peroxidation process via its direct reaction with LH to form LOOH, as well as to cause DNA damage. Since M D A cannot be produced as a direct consequence of singlet oxygen reaction with LH, its production requires further propagation of the peroxidation process via possibly metal ion-catalysts. Ex-
perimcnts arc clearly needed to further clarify the identity of the involved activated species and the molecular paths of UVA-induced lipid l~eroxidation. Owing to the potential involvement of MDA in the formation of lipofuscins [46] and in reaction with DNA [47,52] further studies should be undertaken to confirm the production of M D A in the course of UVA-induced lipid peroxidation. Further attention should be paid to the formation of other peroxidized materials since it is generally considered that MDA is a minor by-product of lipid peroxidation in various peroxidizing systems [531.
References i ('oohil, T.P., Peak, M,J, and Peak, LG. (I'087) Phott~hem. Photobiol. 46, 1043- i',1511. 2 Gangc, R,W. and Rosen, C.F. (!'080) Pholochcra. Pholobiol. 43, 7111-7115, 3 Kantor, G.J. (It}85) Photochem. Photobiol, 41,741-7-16. 4 Moan, I. and Peak, M.J.i.Iq891 J. Pholochem. Photobiol. B: Biol. 4, 21-34_ 5 Peak, M.J. und Peak, J,G, (1080,1 in The Biological Effects of UVA Radialiop (Urbach. F. and Gange, R.W., cds), pp. 42-56. Pracger, Nexx York. 6 Stabcrg, B.[,. and Wulf. |t.C. (lth',I3) J. lnves.:, Dermatol. 81, 5t7-5t0. 7 Van Vclden. H. and Van tier Leun, J.C. (1985; Pholochem. Photobiol. 42, 3119-4(13, S Black, FI.S. 119871 Photochem. Photebk~l 40,, 213-221, t~ TyrrelL R.M, and Keyse, S.M. (19901 J, Phottvchem. Phozobiol, B: Biol. 4, 34o-301. 1o Mo5.~, S,1-1. and Smith, K.C. 11081) Pholochcm. Photobiol, 33, 2t13-210, II Tyrell, R.M. 11979) Acta Biol. Med. Germ. 38, 1!s9-t269. 12 Wcbb, R.B. (It)77)in Photochemistry and PhotobLomg: Reviews (Smilh, K.C., ¢d.), Vol. 2, pp. 16t~-262. Plenum Press, Ne'~ York. 13 Dant)ure, I t.J. and Tyrett, R.M. (1976)Pholochcm. Photobiol. ".~, 171-177. 14 Tyrcll, R.M. und Pidoux, M, 11089) Phott~'hem, Pholobiot. 4~), 407-412,
15 Melteft, t1., Diczel, W. and Sonnichsen, N. (t97h) Experentia 32, 1397-1308. If) Pathak, M.A. ,.nd StraUon, K. (1968) Arch. Biochem. Bit}phys. 123, 4~8-47~,, 17 Chamberlain, J, and Muss, S.H. 11987) Photochcm. Pholobiol. 45, 625-tGI1, IN Pathak, M.A, and Carraro, C. (19851 Photochem. Pholobiol. 4Is, 74. 19 Thomas, J.P. and Girotd, A.W. 11989) Cancer Rcs. 49, 1682-168h. 211 Mandal. T.K. and Chanerjec, S,N. (lqS0) Radial. Res. 83, 2911~
3O2. 21 Ding, A.tl. and Chan. P.C. 110841Lipids, It), 278-284. 22 Girotti, A.W., Thomas. J.P, and Jordan, J.E. (1985) Pholochcm. Photobioi. 41. 2t,7-276, 23 Gironi. A.W. (19~1) Photochem. Photobiof.51,497-509. 24 Tyrcll. R,M. and Pidoux. M. 119881 Pholochcm. Photohiol, 47, 4115-412. 25 Coulomb. B, Saiag, P., Bell, E., Beitburd, F., Lehreton, C., I ; ",lan, M. anti Dubertret, k. (1980)) Br, J. Dermatol. 114, 91 - I l l l .
2fl Lo~,"y, O.H.. Rosebrough. N.J. ;.rod Farr. A.L. 11951) J. Biol.
Chem. 193, 265-275. 27 Aust, S.D. (11186)in Handbook of Methods for Oxygen Radical
268
28
29
30 31 32 33 34 35 36 37 38 39 4f)
Re~,earch (Grennwa!d, R.A., ed), pp. 203-207, CRC Press, Boca Raton. Wong, S.H.Y.. Knight, J.A., Hopfer, S.M.. Zaharia, O.. Leach. C,N.. Jr. and Sunderman, F,W.. Jr. (19871 Clin. Chem. 33, 21422(|. Bergmeyer. H,U, Bernt, E. and Hess. B. tl9b3) in Methods of Enzymatic Analysis (Bermeyer, H,U.. ed). pp. 736-741, Verlag Chemic. Berlin, Black, f-I.S,. Lenger, W,A,. Gerguis. J. and Thornby, J.i. (1985) Cancer Res. 45. 6254-6259. Girotti, A.W. ( 19851 J, Free Rad. Biol. Med. 1, 87-95, Pryor W.A, 11978) Photochem. Photobiol. 28. 787-801. Brown. K. and Fridovich, i. (198I) Arch. Biochem. Biophys. 2(16, 414-4t9, Suunmerfield, F.W. and Tappel, A.L. (19841 Anal. Biochem, 143. 265-27 I. Minotti, G. and Ausl. S.D. (1981) Chem. Phys. I,ipids 44, 191-2(]8. Frankel. E.N, 1,19851 Prog. Lip. Re'.;. 23, 197-22l. Tappel E.N. (19801 in Free Radical in Biology (Pryor, W.A., ed.), Vol. 4. pp. 1-45, Academic Press. New York. Kappus, tt. (19851 in Oxidative Stress (Sies, E., ed). pp. 273-31(I, Academic Press, Orlando, Knight. J.A., Pieper, R.K. and Mc Clellan, L. (19881Ciin. Chem. 34, 2433-2438. Bird, R.P. and Draper, H.H. (19841 Methods Enz3'mol. 105, 299-305.
41 Draper, H.H. and Hadley, M, (199(I) Methods Fnzymol. 186. 421-43 I. 42 Bigwood. T. and Read, O. (1989) Free Rad. Res. Comms. 6. 387-39_.2.
43 Kosugi. H. and Kikugawa, K. (1989) Free Rad. Biol. Med. 7. 205-2(17.
44 Outteridge, J.M.C. and Quinlan, G,J. (19831 J. Applied. Biochem. 5. 293-299. 45 Esterhauer, H. and Cheesman. K. (199111 Methods Enzymol. 186, 4t17-42 I. 46 Tsushida, M., Miura, T, and Airaba, K. (19871 Lipids 4, 297-325. 47 Vaca, C,E.. Wilhelm. J. and Harms-Ringdahl. M. (19881 Mut, Res. t 95, 137-149. 48 Reyftman, J-P,, Kohen. E., Morli~re, P., Santus, R., Kohen, C., Mangel. W.F., Dubertret. L. and Hirscb.berg. J.G. (19861 Photoehem. Photobiol. 461-469. 49 Niki. E. (19871 Chem. Phys. Lipids. 44, 227-253. 50 Hanson, D.L and DeLed, V.A. (19891 Photochem. Photobiol. 49, 423-430. 51 All, M.. Gudbranson. C.O. and McDonald. J,W.D. (19801 Proslagtandins and Medicine 4. 79-85. 52 Mukai, F.H. and Goldstein, B.D. (1976) Science 191,868-869. 53 Halliwelt, B. and Guttridge, J.M.C. (19901 Methods Enzymol. 186, 1-85.
