STUDIES ON THE MODE OF ACTION OF PSORALEN DERIVATIVES I. THEIR EFFECT ON COPPER AND GLUTATHIONE LEVELS IN BLOOD AND LIVER*
ANWAR EL-MOFTY, M.B., C.H.B., M.D., ABDEL MONEM EL-MOFTY, M.B., C.H.B., D.V.D. (lioNs.), M.D., HASSAN ABDELAL, M.B., C.H.B., D.V.D. AND M. F. S. EL-HAWARY, B.Sc. (lioNs.) CAIRo, Pn.D. Psoralens were first introduced into medical use glutathione). They found that treatment with as a treatment for vitiligo 1948 as ammoidin psoralen compounds caused definite rise in serum
(8-methoxypsoralens) and ammidin (5-isoamyl- copper levels of both leukodermic patients and ene oxypsoralen) which were separated from the normal controls. It was therefore thought of interest to explore the extract of fruits of an Egyptian herb Ammi &tajus Linn (1). The efficiency of this group of in detail the action of psoralens on copper metabodrugs promoting melanogenesis has been es- lism and glutathione levels in rats in an attempt tablished by many workers in this field (2—7).
From clinical observations A.M.El-Mofty (8)
(in press) reported that the supplementation of copper as copper sulfate—given orally—to the treatment of vitiligo with psoralen compounds
to determine the mode of action of the psoralens on pigmentation. Analysis of blood and liver for their copper and glutathione contents in experimental animals receiving the psoralen derivates was done and compared with control groups. We
showed great improvement in repigmentation tried to compare the effect of pure crystalline and raised the percentage of improvement and psoralen, ammoidin (8-methoxy psoralen) and complete cure to 47%. ammidin (5-isoamylene oxypsoralen) to study It is well established that under normal con- the effect of each drug separately since the drug ditions the process of melanin formation goes used in treatment was usually a mixture of on steadily as a result of the enzymatic activity psoralen compounds. of tyrosinase, a copper metalo enzyme. In 1951 EXPERIMENTAL Lerner et al. (9) reported that copper essential In this work albino rats weighing from 200 gm. for tyrosinase activity in mammalian tissues inwere used. Ammoidin (8-methoxypsoralen) and cluding human epidermis, and more recently in 1958 Breathnach (10) found that the addition of dilute copper sulphate solution to the tyrosinase solution leads to a speeding up of the action in
the melanocytes of both freckles and pale epi-
dermis. On the other hand it was found that
ammidin (5-isoamylene oxypsoralen) were kindly supplied by Memphis Chemical Company as pure chemical materials. Doses of 800 mgm./Kg. body weight of either drug were used in the brief experiments whereas doses of 300 mgm./Kg. body weight were used in
many organic sulfur compounds such as cysteine the longer experiments. The doses were given and glutathione are active inhibitors of enzymatic orally mixed with food as an emulsion, forced-fed melanin formation in vivoas well as in vitro (11, 9). with a polythene tube attached to a syringe. The
It is thus evident that all factors leading to disturbance in melanin formation could be traced
to an upset of a final balance between copper tyrosinase and the inhibiting-SH group.
A study was carried out by A.M.El-Mofty, Fawzy, El-Hawary (12) to investigate the biochemical effects of psoralen therapy on the two important factors controlling tyrosinase activity, namely, the concentration and availability of copper and—SH group (represented mainly as * From the Departments of Medicine and Dermatology, Cairo University, Cairo, Egypt, and
experimental period for the short experiments was 24—48 hours and for the longer experiments it was 16 days.
The life span of albino rat is assumed to be 3 years. This may be considered as the equivalant of 90 years of human life (19). Sixteen days in
the life span of albino rats can be considered equal to 16 months of human life.
At the end of the experimental period the animals were sacrificed by decapitation. The blood
was collected from the neck; and the animal was immediately dissected and tissue (liver) needed for experimentation was quickly removed,
the Serum and Vaccine Institute, Biochemistry washed in physiological saline, blotted against filter paper to remove any extra fluid, then weighed
Research Unit, Agouza, Cairo. Received for publication September 19, 1958.
and kept in the deep freeze until it was analyzed. 645
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TABLE I Tissue copper and glutathione contents in normal rats Results are expressed as mg. glutathione/100
ammoidin in doses of 800 mg/Kg. body weight causes a dramatic increase in blood copper, a
ml and as microgram Cu/l00 ml in blood and
/100 gm. fresh weight in case of tissues.
decrease in the liver copper content from a normal
normal mean value of 131 to mean value of 325
associated with a marked
mean value of 113 to 37 mierogram/100 gm.
Rat No. 1
These figures show that administration of
fresh tissue weight.
The results are graphically represented in Fig. 1.
Regarding the effect of brief experimentation with ammoidin on tissue glutathione, the figures
show a marked decrease in liver glutathione content from a normal mean value of 94 to 48
21.2 22.2 24.6 24.3 23.5 26.8 23.8
G.S.H. Liver 109 G.S.H.
mg./100 gm. tissue fresh weight;whereas in blood,
79.6 83.7 96
98.3 87.4 94
the decrease was not great (only from a normal mean value of 23.8 to a mean value of 20.4 mg. glutathione/100 ml. blood). Yet in some rats (No. 1, 2, 6) the decrease in the levels of blood gluta-
The method for estimation of blood glutathione, worked out by Thompson and Watson in 1952 (13), was adopted in this work. Tissue glutathione was estimated by the method of El-Hawary (14).
The method of Gubler, G. J. et at. (15) for the
estimation of serum copper was followed.
thione is significant.
The results obtained from blood and liver copper and glutathione content in rats receiving
doses of 800 mgm. ammidin/Kg. body weight in the short experiments are given in Table III.
These figures show that the administration of
ammidin in the previously mentioned doses has
For the estimation of tissue copper content the no appreciable effect on copper and glutathione same procedure was applied using tissues homocontent of blood and liver. genates prepared as follows: It was thought that the results obtained in About one gm. of the liver was homogenized the short experiment may not represent the and made up to a volume of 4.5 ml. Three ml. of this homogenate were transfered to a conical cen- changes occurring in treating the patients with trifuge tube (capacity 15 ml.). To it was added 3 theraputie doses of psoralen compounds. For ml. of 2N HC1. The contents were mixed well and this reason we carried out some chronic experileft to stand for 10 minutes. Then 3 ml. of 20% ments using sublethal doses of ammoidin, 300 trichioro acetic acid solution were added, the con- mgm./Kg. body weight daily for 16 days. The tents again mixed well and left to stand for 10 results of these prolonged experiments are repreminutes. The tubes were then centrifuged for 30 sented in Table J1/ minutes at 300 r.p.m. Two and a half microgram of the supernatant-fluid were used for the deterTABLE II mination of copper content following the same Liver Blood procedure of Cubler et at. (15). Rat No. RESULTS
The results obtained for the different rats are
Table I shows the values obtained for tissue
copper and glutathione contents in rats receiving no drugs.
The results obtained for blood and tissue
glutathione and copper content in rats receiving doses of 800 mg. ammoidin/Kg. body weight in the short experiments are given in Table II.
noted in the following tables.
Mean S .E.
MODE OF ACTION OF PSORALEN DERIVATIVES Liver Copper
300 280 260 240
220 200 180 160
60 40 20
Fio. 1. Effect of Ammoidin on copper levels. Rat TABLE IV
The Effect of Ajumidin on
No. of Rats No. of Rats
4 5 6 Mean S.E.
18.8 21.2 21.2 20.3 18.4 20.5 19.7
112 108 115 113 112
72.4 68.2 74.6 73.4 71.2 74.6 72.4
110 121 101 103
2 3 4
29.2 24.3 30.8 25.2
315 344 275 357 325
is again a huge rise in blood coppr from a normal
From the data given in Table IV it is clear mean value of 131 to a mean value of 341 microthat prolonged doses of ammoidin (8-methoxy- gram%, and this was associated with a decrease psoralen) have almost the same effect as the in the level of liver copper from normal value of doses of the shorter ammoidin treatment. There 115 to 84.5 mg./100 gm. tissue fresh weight. The
THE TOURNAL OF INVEBTIGATIVE DERMATOLOGY Blood Copper
280 260 240 220 200 180 160 140 131
80 60 40 20 0 FIG. 2
latter decrease is less dramatic than the decrease amylene oxypsoralen) is completely inert and occurring in the short ammoidin course of treat- has no effect at all on copper or glutathione levels in blood or liver. ment. On the other hand, the effect of prolonged With regard to the effect on —811 group, it doses of ammoidin on blood and tissue glutathi- appears that the blood changes are not striking. one is less than the corresponding effect in If the changes are compared to changes in blood the shorter course dosage. copper one is tempted to relate the therapeutic The effect of ammoidin in prolonged doses on value to its effect on blood copper rather than its tissue and blood copper and glutathione is effect on —Sil group. graphically represented in Fig. 2. The explanation of hepatic depletion of —511 group under the effect of psoralens is not clear. DISCUSSION
We are tempted to suggest that the cause is The findings show beyond any reasonable disruption of glutathione rather than its mobi-
doubt that ammoidin (8-methoxy psoralen) is a strong hyper-cupremic agent and that it leads lization from liver since no striking elevation of to elevation of blood copper through mobilization —SU level in the blood was obtained. The doses used in both experiments are far above maximum of liver stores of copper. This was demonstrated in both the short and therapeutic effective doses. This was intended to the prolonged experiments. Ammidin (5-iso- accentuate the changes for purposes of demon-
RODE OF ACTION OF PSORALEN DERIVATIVES
stration. The theraputic doses in man (12) lead by Memphis Chemical Co., Cairo, Egypt to whom to definite rise in blood copper levels. The mechanism by which psoralens mobilize
copper from liver to blood was in urgent need of investigation not only to elucidate the mode of action, but also to exclude the possibility that copper is mobilized as a result of a direct toxic effect on the liver by the drug. The results are going to be reported in the following paper. But
it suffices here to report that the drug loses completely its mobilizing effect on copper (from
liver to blood) in hypophysectomized animals. This excludes completely a direct toxic effect on the liver, causing cellular disruption and mobilization and escape of copper to blood. Keeping in mind the importance of copper and copper tyrosinase in melanogenesis, the elevation
of blood copper under ammoidin might throw light on its mode of action and its therapeutic
we are grateful.
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