The effect of griseofulvin on the heme pathway. Studies on tissue explant cultures

Toxicology, 46 (1987) 73--82 Elsevier Scientific Publishers Ireland Ltd.

THE EFFECT OF GRISEOFULVIN ON THE HEME PATHWAY. STUDIES ON TISSUE EXPLANT CULTURES*

ELBA VAZQUEZ, SUSANA AFONSO, ANA MARIA BUZALEH, NORA NAVONE, CESAR POLO, ERNESTO SCHOUA and ALCIRA BATLLE Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP) - F C E N (UBA} and CONICET, Ciudad Universitaria, Pabell~n II, 2do. Piso, 1428 Buenos Aires (Argentina)

{Received January 9th, 1987) (Accepted April 10th, 1987}

SUMMARY

The effect of griseofulvin on porphyrin biosynthesis was studied in vitro and in vivo using liver and skin explants from mice. Neither porphyrin nor precursors accumulation was detected after in vitro treatment of explants with griseofulvin. Culture medium porphyrins formed from added d-aminolaevulinic acid (ALA) were increased after oral chronic intoxication. Similar results were obtained by cutaneous and oral intoxication. Consequently griseofulvin topical application is proposed as an alternative method to produce experimental erythropoietic protoporphyria. Probable intoxication mechanisms are proposed to explain the porphyrinogenic action of griseofulvin.

Key words: Griseofulvin; Haem; Porphyrins; N-methylporphyrins; Tissue culture INTRODUCTION

In erythropoietic protoporphyria {EPP), protoporphyrin (Proto) accumulates in large m o u n t s , particularly in the liver [1--6] and erythrocytes [7], reflecting a deficiency of protohaem ferrolyase {EC 4.99.1.1.} activity [8--10] {Fig. 1). The most frequently occurring symptom is increased cutaneous *Dedicated to Professor Claude Rimington, FRS on the occasion of his 85th birthday. Address all correspondence and reprint requests to: Prof. A. BatHe, Viamonte 1881 10 A, 1056 Buenos Aires, Argentina. Abbreviations: ALA, 6-Amlnolaevulinic acid; Copro, coproporphyrin; EPP, erythropoietic protoporphyria; Gris, griseofulvin; PBG, porphobilinogen; ProW, protoporphyrin; Uro, uroporphyrin. 0300-483X/87/$03.50

© 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in ~reland

73

HAEM

GLYCIN

÷

SUCCINYL CQA

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CYTOPLASM

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.

.

.

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.

.

.

.

.

.

!~ED B ! 2 0 0

PBG

"--.. /

COPROgen

CEL.~ F~ECLS •

UROgen

Fig. 1. Enzymatic defect in EPP.

photosensitivity [7,11] which results from the interaction of cellular structures with highly reactive singlet oxygen generated by energy transfer from photoexcited triplet state Proto in skin [12,13]. Proto accumulates in lipophylic environments [14] and the resulting phot~damage would affect lipid-contalning structures [15]. Adding exogenous Proto, it was demonstrated that the damage to plasma membranes and mitochondria occurs earlier than the photo, damage to lysosomes [16]. Nevertheless, it is not known yet, whether Proto in the skin of E P P is generated locally [17] or is accumulated from Proto coming from plasma [18]. A suitable model simulating human E P P is developed by administration griseofulvin (Gris) to animals, which produces in mice liver accumulation of Proto [19,20] with liver damage [21] and increased cutaneous photosensitivity [19]. It has been suggested that the last effect would be promoted by Proto circulating in the serum after long-wave U V light radiation [22]. However,-high levels of Proto (1300 nmol/g wet wt) and uncoupling of oxidative phosphorylation in the liver of Gris-intoxicated mice were found even without exposure to light [15]. These findings suggest, that liver failure in Gris-induced protoporphyria and in EPP, m a y result from mitochondrial disfunction. It has been shown that the porphyrinogenic effect of Gris is mediated through N-methylporphyrins accumulation which inhibit protohaem ferrolyase activity [23--28], attributed to a strong interaction between the modified porphyrin and the enzyme active center [24]. It is important to note that the Proto alkyl derivates or certain N-alkyl-mesoporphyrins are also powerful inhibitors of protohaem ferrolyase [25]. W e have developed a tissue explant culture system, with the purpose of investigating the origin of skin porphyrins, the cutaneous toxicity caused by certain drugs as well as their mechanism of action [29]. Using this experimental model, we decided to investigate the in vivo and in vitro effect of Gris on haem biosynthesis in mice liver and skin explants.

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MATERIALS AND METHODS

Animals Male mice {Mus musculus strain OF 1) housed in controlled climate conditions with free access to normal laboratory animal feed and water were used.

Intoxication The animals received 5 rag/mouse per day (chronic intoxication) or 50 mg/ mouse per day {acute intoxication} of Gris (Grisovin F.P. 500 MR, Glaxo) in the drinlelng water, Gris was previously dissolved in ethanol {12.5 mg/ml ethanol or 125 mg/ml ethanol]; control animals received only ethanol/water in the same final proportion (1 : 5). For cutaneous intoxication, the Gris was dissolved in olive off (500 nag Gris/10 ml oil}, using a wool sprinkler for topical application on the shaved back skin, applicated every 48 h during 1 week. Tissues

Thorax and abdominal skin bands of the mice were carefully shaved post mortem and washed by spraying with 70% ethanol. The subcutaneous fat was removed and the skin was cut in small pieces {approx. 40 mg), washed with sterile isotonic saline solution, dried, weighed and kept at 0°C until employed {approx. 15 rain). The animal was perfused with sterile saline solution before removing the liver and the tissue was treated as described for skin samples, using pieces of approximately 100 mg each. Aseptic conditions were maintained during explants preparation.

Culture chamber The culture chamber was a modification of that originally designed by Fell and Robison [30] {Fig. 2). The explants were placed on stainless steel supports [29]. In all the experiments cultures were incubated at 37 °C during 24 h in darkness, as previously described [29]. The culture medium was a balanced salt solution (NaC1 6.800 g/l; KC1 0.4 g/l; CaCl 2 • 2 H~O 0.264 g/l; MgSO 4 . 7 H 2 0 0.200 g/l; NaH2PO 4 • 2H20 0.140 g/l; NaHCO 3 1.680 gfl and glucose 1.000 g/l} with the addition of chloramphenicol (1.6 rag/dish). All the measurements were made on the culture medium.

ALA and porphobilinogen (PBG) determination A L A and P B G were determined by the method described by Mauzerall and Granick [31].

Determination of porphyrins Porphyrins were measured following the method described by With [32].

Porphyrin chromatography Porphyrin chromatography was described by Afonso et al. [33].

performed according to the method

75

Wet ~otton

Tissue 1 /

J

Culture Medium

Stainless

Steer

9 upp°rt \

Petri Dish

Fig. 2. Culture chamber.

Determination of liver endogenous porphyrins Endogenous porphyrins were determinated in a fiver homogenate preparation according to Afonso et al. [33]. RESULTS

In order to s t u d y the in vitro and in vivo effect of Gris on the haem pathway, mouse fiver and skin explants in culture were used, adding only ALA (0.8 rag/dish) [29] as substrate with or without Gris in the medium. Precursors and porphyrins were determined on the maintenance medium after the incubation period. It was assumed that all the porphyrins accumulated in the medium were formed from the exogenous A L A added to the dish, since the amount of porphyrins measured when the tissues were incubated without substrate was negligible. In spite of this, blanks were always run in each assay. In vitro treatment To determine whether Gris has only local action at skin level in vitro, different amounts of this toxic were added to the maintenance medium of explants. The Gris was previously dissolved in ethanol, in an appropiated concentration so that only 0.1 ml of solution was added to the dish (total vol. = 50 ml); a dish containing only ethanol (0.1 ml) in the medium was used as control. Table I shows the results obtained. Neither precursor consumption nor porphyrin accumulation was significantly different as compared to the control values obtained when the explant was incubated without adding Gris. On the other hand, medium porphyrin accumulation was increased compared to control values, when liver explants were treated in vitro under the same experimental design (data not shown}. In vivo treatment Since Gris added to the maintenance medium apparently produced no effect on porphyrin biosynthesis, the action of both oral and cutaneous intoxication in animals was investigated. Oral chronic intoxicatio,~ Gris oral administration effect on porphyrin

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TABLE I E F F E C T OF G R I S A D D E D TO T H E C U L T U R E M E D I U M

G R I S added (rag/dish)

A L A consumed {~g/mg tissue)b

P B G consumed" (~g/mg tissue)b

Porphyrins accumulation (ng/mg tissue)b

0.1 1.0 Control

14.0 ± 2.6 c 11.0 ± 1.4 c 12.0 ± 2.0

9.1 ± 1.6 c 7.2 ± 0.4 c 7.7 ± 0.8

1.23 ± 0.13 c 1.72 ± 0.08 c 1.50 ± 0.18

a PBG consumed was theoretically calculated taking into account the PBG formed from the A L A consumed and the remaining PBG. Culture conditions are given in the text. b Each value represents the mean ± S.D. of experiments in which tissue from 5 animals were used. c Not significantly different as compared to untreated controls.

accumulation was studied using skin and liver explants. Liver was used as a comparative parRmeter in order to evaluate the intoxication degree. A linear increasing relationship between porphyrin accumulation and intoxication time was observed with both tissues (Fig. 3). The longer the intoxication time elapsed, the higher the proportion of less carboxylated porphyrins observed, when chromatographic analysis was carried out on samples from liver, instead in skin culture medium, only uro-porphyrin (Uro) was detected. Cutaneous intoxication. Gris was topically applied every 48 h during a week with the aim of studying the kind of response produced as a result of the different route of administering the toxic. An increase of about 100% in the liver and skin porphyrin accumulation as compared to the control value

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SKIN

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Fig. 3. Effect of chronic oral intoxication with Gris on porphyrin biosynthesis by liver and skin explants. Experimental conditions were as described in Materials and Methods. (O) control. ( I ) intoxicated. Each value represents the mean of experiments in which tissue from 5 animals were used.

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TABLE I I VIA CUTANEA INTOXICATION Tissue

Porphyrins accumulated {ng/mg tissue)a

Control

Liver Skin

3.10 ± 0.14 1.80 ± 0.34

Treated

Liver Skin

6.30 ± 1.21 b 4.50 ± 1.02 b

a Each value represents the m e a n ± S.D. of experiments in which tissue from 5 animals were used. b p < 0.01 by Student's t-test in comparison to controls.

was obtained {Table II). The chromatographic analysis revealed the presence of all the tetrapyrrolic intermediates up to coproporphyrin (Copro), but in 1 case Proto was detected. Oral acute intoxicatior~ Simultaneously to the cutaneous intoxication experiments described previously, an acute oral intoxication was carried out during the same period. The results are shown in Table III. In liver, porphyrin accumulation increased about 200%, while in skin no difference was detected with respect to the controls. Comparing these results with the values obtained after cutaneous Gris administration {Table II), it is clear that the effect produced in liver is the same despite the route of administration; however, the skin was only affected during the oral chronic or cutaneous intoxication.

Endogenous porphyrin accumulation To establish whether the manner of intoxication would produce any effect in total porphyrin accumulation in the liver, endogenous porphyrins were TABLE Ill ACUTE ORAL INTOXICATION Tissue

Porphyrins accumulated (ng/mg tissue~a

Control

Liver Skin

3.10 ± 0.20 2.30 ± 0.46

Treated

Liver Skin

8.50 ± 1.64 b 2.40 ± 0.60c

a E a c h v a l u e r e p r e s e n t s t h e m e a n _+ S.D. of e x p e r i m e n t s in w h i c h t i s s u e from 5 a n i m a l s were used. b p < 0.01 b y S t u d e n t ' s t - t e s t in c o m p a r i s o n to controls. c N o t s i g n i f i c a n t l y different as c o m p a r e d to u n t r e a t e d controls.

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T A B L E IV E N D O G E N O U S P O R P H Y R I N S IN L I V E R A n i m a l s were killed 1 week after dosing. E n d o g e n o u s p o r p h y r i n s (pg/g tissue}a Control Via c u t a n e a Oral way

8.2 ± 2.2 10.6 ± 2.0

Treated 34.5 ±

4.6 b

70.5 ± 12.1 b'¢

° E a c h value r e p r e s e n t s t h e m e a n ± S.D. of e x p e r i m e n t s in which tissue from 5 animals were used. b io ~ 0.01 b y S t u d e n t ' s t-test in comparison to controls. e P < 0.01 by S t u d e n t ' s t-test in comparison to via c u t a n e a t r e a t m e n t .

measured in a liver homogenate. Table IV shows the results obtained for liver explante of mice orally, acute or cutaneously intoxicated. In the former the endogenous porphyrins were 100% higher than the values detected in the latter. Nevertheless, in both cases, the endogenous porphyrin accumulation was significantly increased with respect to the control animals. DISCUSSION

De Matteis and Gibbs [34] have reported that Gris and isogriseofulvin promote accumulation in the liver of a pigment with inhibitory activity towards protohaem ferrolyase. Other pigments have been reported to accumulate, after administration of drugs containing unsaturated side chains, but they do not appear to inhibit this enzyme [25]. These pigments are modified porphyrins with an abnormal substituent at one of their pyrrole nitrogen atoms and their differential behaviour on protohaem ferrolyase activity could be due to dissimilarity in the nature and size of substituent [24,25[. We have now demonstrated that Gris added in vitro has no effect on porphyrin p a t h w a y in skin explants {Table I}, however after in vivo treatment, an inhibitory action was revealed which was dependent on the route of Gris administration although we can not yet offer a good explanation for this phenomena. In chronical oral intoxication, increased porphyrin accumulation was observed both in skin and liver maintenance medium {Fig. 2}. The same response was found in the cutaneous intoxication {Table IIL This toxic effect on haem metabolism was corroborated by an enhancement in the liver endogenous porphyrin accumulation {Table IV}. Talring into account these results, it is proposed that Gris cutaneous administration is an alternative method to produce an experimental EPP.

79

In oral acute intoxication a great accumulation of porphyrins was observed in the liver maintenance medium, while in skin no differences were found with respect to the control values {Table III}. On the basis of these findings, the following mechanisms to explain the toxicity of Gris are postulated: 1. Mechanism proposed for the intoxication process via cutanea. An endogenous porphyrin accumulation increase is evident. Plasma

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In this mechanism the total functionality of the 'liver-plasma-skin' system is necessary to obtain intoxication by Gris in skin. It could probably be due to its inability to synthesize per se N-methyl-porphyrins. These compounds would be formed in the liver and then transported by blood stream to skin, where they would inhibit ferrolyase. It is possible that a small molecular weight endogenous compound with methylating capability (methylating factor, MF) [28], already formed in the liver, would be transported to skin and in this tissue would bind to the N-pyrrolic atom of porphyrins inducing the N-methyl-porphyrin formation. ACKNOWLEDGEMENTS

Grants from the National Research Council (CONICET), the SUBCYT, University of Buenos Aires, Secretaria de Salud Pfiblica and Banco de la Naci6n Argentina are acknowledged. Alcira M. del C. Batlle holds the post of Principle Scientific Researcher at the CONICET. Elba Vfizquez, Susana Afonso, Ana Marfa Buzaleh, Nora Navone, C6sar Polo and Ernesto Schoua are Research Fellows at the CONICET. The valuable help of Rodolfo Biekofsky, Laura Dada and Jorge Muschietti in some experiments is greatly appreciated.

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