Chemoprevention of smoke-induced alopecia in mice by oral administration of L-cystine and vitamin B6 Francesco D’Agostini a, * , Paolo Fiallo b , Tanya M. Pennisi a , Silvio De Flora a a Section of Hygiene and Preventive Medicine, Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy b Section of Social Dermatology, Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy Received 7 September 2006; received in revised form 14 February 2007; accepted 19 February 2007 Journal of Dermatological Science (2007) 46, 189—198 www.intl.elsevierhealth.com/journals/jods KEYWORDS Cigarette smoke; Alopecia; Apoptosis; Chemoprevention; L-Cystine; Vitamin B6 Summary Background: We previously demonstrated that high doses of environmental cigarette smoke (ECS) induce alopecia in mice. This effect was prevented by the oral admin- istration of N-acetylcysteine (NAC), an analogue and precursor of L-cysteine and reduced glutathione. Objectives: The present study aimed at assessing whether L-cystine, the oxidized form of L-cysteine, which is a key hair component, may behave like NAC in inhibiting ECS-induced alopecia and modulating the mechanisms responsible for this condition. Methods: C57BL/6 mice were exposed whole-body to ECS in a smoking machine. Groups of mice received in the diet, at three dose levels, a mixture of L-cystine with vitamin B6, which plays a role in L-cystine incorporation in hair cells. Occurrence of alopecia areas and apoptosis of hair bulb cells were evaluated for up to 6 months of exposure, and the time course induction of micronucleated erythrocytes in peripheral blood was investigated. Results: The frequency of micronucleated erythrocytes was increased by ECS, irrespective of treatment with L-cystine/vitamin B6. ECS-induced alopecia and apoptosis of hair bulb cells in all exposed mice. L-Cystine/vitamin B6 inhibited alopecia in a dose-dependent fashion. Conclusions: High-dose ECS induces apoptosis-related alopecia in mice, and oral administration of L-cystine/vitamin B6 is an effective preventive treatment. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +39 010 353 8487; fax: +39 010 353 8504. E-mail address: [email protected](F. D’Agostini). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.02.005
Transcript
Chemoprevention of smoke-induced alopecia inmice by oral administration of L-cystine andvitamin B6
Francesco D’Agostini a,*, Paolo Fiallo b, Tanya M. Pennisi a, Silvio De Flora a
a Section of Hygiene and Preventive Medicine, Department of Health Sciences, University of Genoa,Via A. Pastore 1, I-16132 Genoa, Italyb Section of Social Dermatology, Department of Health Sciences, University of Genoa,Via A. Pastore 1, I-16132 Genoa, Italy
Received 7 September 2006; received in revised form 14 February 2007; accepted 19 February 2007
Journal of Dermatological Science (2007) 46, 189—198
Background: We previously demonstrated that high doses of environmental cigarettesmoke (ECS) induce alopecia in mice. This effect was prevented by the oral admin-istration of N-acetylcysteine (NAC), an analogue and precursor of L-cysteine andreduced glutathione.Objectives: The present study aimed at assessing whether L-cystine, the oxidizedform of L-cysteine, which is a key hair component, may behave like NAC in inhibitingECS-induced alopecia and modulating the mechanisms responsible for this condition.Methods: C57BL/6 mice were exposed whole-body to ECS in a smoking machine.Groups of mice received in the diet, at three dose levels, a mixture of L-cystine withvitamin B6, which plays a role in L-cystine incorporation in hair cells. Occurrence ofalopecia areas and apoptosis of hair bulb cells were evaluated for up to 6 months ofexposure, and the time course induction of micronucleated erythrocytes in peripheralblood was investigated.Results: The frequency of micronucleated erythrocytes was increased by ECS,irrespective of treatment with L-cystine/vitamin B6. ECS-induced alopecia andapoptosis of hair bulb cells in all exposed mice. L-Cystine/vitamin B6 inhibitedalopecia in a dose-dependent fashion.Conclusions: High-dose ECS induces apoptosis-related alopecia in mice, and oraladministration of L-cystine/vitamin B6 is an effective preventive treatment.# 2007 Japanese Society for Investigative Dermatology. Published by Elsevier IrelandLtd. All rights reserved.
0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jdermsci.2007.02.005
Cigarette smoke (CS) is a major risk factor for thosechronic degenerative diseases that are the leadingcauses of death in the population, such as cardio-vascular diseases, cancer, chronic obstructive pul-monary diseases, etc. [1,2]. The involvement oftobacco smoking has also been proved in a varietyof skin diseases and dermatological conditions, suchas poor wound healing, wrinkling and prematureskin aging, squamous cell carcinoma, psoriasis,hidradenitis suppurativa [3]. In addition, thehypothesis has been raised that smoking may beassociated with hair loss in humans [4,5].
We previously demonstrated that a heavy whole-body exposure to environmental cigarette smoke(ECS) is capable of inducing alopecia in C57BL/6mice [6,7]. The ECS-induced alopecia, as well as hairloss induced in mice by intravenous administrationof the cytotoxic drug doxorubicin, could be effi-ciently prevented by oral administration of the thiolN-acetylcysteine (NAC) with the drinking water[6,8]. NAC is an analogue and precursor of reducedglutathione (GSH), and works mainly as a free radi-cal scavenger and nucleophile. GSH can be used asan antitoxic drug but, being a tripeptide, does noteasily cross cell membranes. L-Cysteine is the rate-limiting amino acid in the de novo synthesis of GSH,but its use in humans is precluded by its toxicity.NAC, whose safety in humans has been documentedin 40 years of clinical use, even at very high doses, isreadily deacetylated when entering cells, therebyincreasing intracellular L-cysteine and GSH stores. Inaddition, NAC works per se, as a sulfur-containingmolecule [9].
The role of sulfur-containing amino acids in hairmetabolism has been reviewed [10]. Since L-cysteine and its oxidized form L-cystine are inter-convertible, we were interested to evaluatewhether L-cystine, which is not toxic and is a keycomponent in hair [11], may behave like NAC ininhibiting hair loss due to toxic agents. A pilot studyin humans demonstrated that a long-term oraladministration of L-cystine may have beneficialeffects on diffuse hair loss [12], and oral prepara-tions containing L-cystine are among suggested cur-rent therapeutic recommendations for hair loss[13,14]. In our knowledge, however, L-cystine hasnever been tested with respect to CS-related hairloss or other adverse effects of CS.
Since L-cystine has been shown to be reduceddramatically in the hair of vitamin B6-deficient rats[15], we decided to combine vitamin B6 withL-cystine. This combination is also justified by thefact that vitamin B6 is important for skin develop-ment and maintenance [16], and the parenteral
administration of vitamin B6 has been associatedwith an improvement in the hair conditions and areduction of hair loss [17].
The results of the present study show that amixture of L-cystine and vitamin B6 is capable ofinhibiting alopecia, in a dose-dependent manner, inthe same C57BL/6 mouse model used for evaluatingthe protective effects of NAC against ECS-inducedalopecia. We provide also evidence that this kind ofalopecia is associated with apoptosis of hair bulbcells.
2. Materials and methods
2.1. Animals
A total of 48 adult female C57BL/6 mice (Morini, S.Polo d’Enza, Italy), aged 8 weeks and having anaverage weight of approximately 20 g, were used.They were housed in Makrolon cages and acclima-tized for a week before starting the treatments. Thetemperature of the animal room was 23 � 2 8C, witha relative humidity of 55%, a ventilation accountingfor 15—20 air renewal cycles/h, and a 12-h day—night cycle. Standard rodent diet (MIL, Morini) wasused. The animals received drinking water ad libi-tum. The amount of food consumed daily by theanimals was measured.
Housing and all treatments of animals were inaccordance with the Italian (D.L. No. 116/92) andEuropean Community guidelines (86/609/EEC Direc-tive).
2.2. Chemicals
L-Cystine and vitamin B6 (pyridoxine chlorhydrate)were supplied by Laboratories Bailleul (Paris,France). The two agents were incorporated in thefood bymeans of a mixer (Blender HB4, Hobart, Troy,OH, USA), adjusting their concentrations as relatedto the body weight of animals and to the daily foodconsumption, in order to obtain a ‘‘calculated dailyintake’’. The highest doses tested were chosen aftera review of the literature [18—22], taking intoaccount toxicity data regarding the two drugs andtheir safe doses recommended for use in humans.
Treatment with these agents started 3 daysbefore the first exposure to CS and continued dailyuntil the end of the experiment.
2.3. Treatments
After a 1-week acclimatization, the mice weredivided into the following experimental groups,each composed of eight animals:
Chemoprevention of smoke-induced alopecia in mice 191
(a) u
ntreated (sham-exposed) mice, kept in filteredair;
(b) u
nexposedmice treated with L-cystine (1000 mg/kg b.w./day) and vitamin B6 (100 mg/kg b.w./day);
(c) E
CS-exposed mice; (d) E CS-exposed mice treated with L-cystine (1000
mg/kg b.w./day) and vitamin B6 (100 mg/kgb.w./day);
(e) E
CS-exposed mice treated with L-cystine(200 mg/kg b.w./day) and vitamin B6 (20 mg/kg b.w./day);
(f) E
CS-exposed mice treated with L-cystine(40 mg/kg b.w./day) and vitamin B6 (4 mg/kgb.w./day).
2.4. Exposure to cigarette smoke
The mice belonging to groups (c)—(f) were exposedto environmental cigarette smoke (ECS) by means ofan automatic smoking machine (mod. TE10-a, Tea-gue Enterprises Inc., Davis, USA) and exposure sys-tem, adjusted to produce a mixture of sidestreamsmoke (89%) and mainstream smoke (11%).
1R3 reference cigarettes (Kentucky Tobacco R&DCenter, University of Kentucky, Lexington, KY,USA), having a declared content of 27.1 mg totalparticulate matter, 22.8 mg tar, and 1.46 mg nico-tine each, were used. Before use, the cigaretteswere kept for 48 h in a standard atmosphere humi-dified with a mixture of 70% glycerol and 30% water.Five cigarettes at one time were burned in themachine, with standard puffs of 35 ml volumeand 2-s duration each. The total particulate matter(TPM) in the exposure chambers was, on an aver-age, 85 mg/m3, and the average CO concentrationwas 350 ppm.
The position of the cages in the exposure cham-bers was rotated daily. The animals were exposed6 h/day, divided into two 3-h rounds with a 3-hinterval. The mice belonging to groups (a) and(b), serving as controls, were kept in filtered airunder identical conditions for the same period oftime.
2.5. Inspection of animals, body weightand survival
The animals were inspected daily for generalaspect, toxicity signs and adverse effects conse-quent to exposure to ECS and/or administration ofthe putative chemopreventive agents. Individualbody weights were registered upon arrival of ani-mals and at monthly intervals, until the end of theexperiment. Survival of mice was also recorded.
2.6. Cytogenetical monitoring
At periodical intervals, i.e., at time 0 and after 7, 14and 28 days of exposure to ECS, samples of bloodwere collected from the tail lateral vein of all mice.Duplicate smears of peripheral blood were stainedwith May-Grunwald-Giemsa. A total of 1000 normo-chromatic erythrocytes (NCE) per slide were scoredand the number of micronucleated (MN) cells wasrecorded. The results were expressed as MN fre-quency/1000 NCE.
2.7. Alopecia
The mice were inspected daily for the presence ofcutaneous and/or hair alterations, alopecia orgreying areas. The frequency of mice with alope-cia was recorded at weekly intervals. All animalswere photographed. The final size of alopeciaareas was measured with the aid of an imageanalysis software (ImageJ, NIMH/NIH, Bethesda,MD, USA).
2.8. Histopathology and apoptosisanalyses
After 6 months of exposure to ECS, all animalswere anesthetized with diethyl ether and killed bycervical dislocation. A complete necropsy of eachmouse was performed. Representative samples ofskin from both healthy areas and alopeciaareas were collected, fixed in formalin andembedded in paraffin. Sections were stained withhematoxylin/eosin for histopathological analysis.Ten sections per mouse were analyzed and thefrequency of mitotic figures in hair follicles wasrecorded.
Apoptosis in hair follicles was evaluated in 5-mm skin sections by TdT-mediated dUTP nickend labeling (TUNEL) method, using a commer-cially available kit (Roche Diagnostics, Monza,Italy) according to the manufacturer’s instruc-tions. The slides were scored at a magnificationof 400�.
2.9. Statistical analyses
Comparisons of treatment-related differencesregarding body weight, frequency of MN and fre-quency of mitotic figures, expressed as mean-s � S.E. within each experimental group, weremade by Student’s t-test for unpaired dataand ANOVA. Comparisons regarding frequency ofalopecia were made by x2 analysis. Differenceswith P < 0.05 were taken as statistically signifi-cant.
192 F. D’Agostini et al.
Table
1Bodyweight(g)ofC57
BL/
6mice(m
ean�
S.E.)
asrelatedto
treatmentan
dtimeoftheexp
eriment
Treatment
Tim
e(m
onths)
01
23
45
6
(a)Controls
22.22�
0.54
22.42�
0.48
22.92�
0.26
22.97�
0.26
23.20�
0.23
23.22�
0.25
23.39�
0.22
(b)
L-Cystine/v
itam
inB6
(100
0/10
0mg/
kgb.w./day
)21
.40�
0.36
22.01�
0.22
22.39�
0.15
22.47�
0.11
23.08�
0.19
23.09�
0.10
23.15�
0.16
(c)ECS
21.41�
0.53
21.10�
0.32
*20
.90�
0.36
***
20.88�
0.35
***
20.79�
0.31
***
20.80�
0.35
***
20.52�
0.28
***
(d)ECS+
L-cystine/v
itam
inB6
(100
0/10
0mg/
kgb.w./day
)21
.74�
0.82
21.27�
0.67
21.34�
0.68
*21
.25�
0.66
*21
.21�
0.63
**21
.32�
0.61
**21
.17�
0.49
**
(e)ECS+
L-cystine/v
itam
inB6
(200
/20mg/
kgb.w
./day
)22
.44�
0.42
21.66�
0.32
21.63�
0.30
***
21.44�
0.28
**21
.57�
0.29
***
21.39�
0.28
***
21.29�
0.29
***
(f)ECS+
L-cystine/v
itam
inB6
(40/
4mg/
kgb.w./day
)21
.25�
0.72
21.07�
0.63
21.04�
0.61
**21
.18�
0.53
**21
.06�
0.48
**20
.91�
0.43
***
20.74�
0.47
***
Statistica
lan
alysis:*P<
0.05
,**P<
0.01
,an
d***P<
0.00
1,as
comparedto
controls.
3. Results
3.1. Survival of mice
All sham-exposed animals belonging to groups (a)and (b) survived after 6 months of treatment. Ingroup (c) one mouse died after 20 weeks of expo-sure to ECS and another one died after 24 weeks.In both groups (d) and (e) one mouse died after 26weeks of exposure to ECS. In group (f) one mousedied after 24 weeks of exposure to ECS andanother one died after 26 weeks. None ofthese differences in survival was statistically sig-nificant.
No macroscopically visible lesion was detected ininternal organs of mice, either spontaneously deador sacrificed after 6 months.
3.2. Body weight
Table 1 reports the body weights recorded in micebelonging to each experimental group (mean-s � S.E.), as measured at the start of the experi-ment (time 0) and, later on, at monthly intervals,until the end of the experiment, immediately beforesacrificing all surviving mice. After 1 month of treat-ment, the body weight significantly decreased in themice exposed to ECS belonging to group (c). Frommonth 2 to the end of the experiment, all animalsexposed to ECS, irrespective of treatment with L-cystine/vitamin B6, belonging to groups (c)—(f),showed a statistically significant loss of bodyweight, as compared to sham-exposed mice. Nosignificant difference could be observed amongstexperimental groups of mice exposed to ECS, asrelated to oral administration of L-cystine/vitaminB6.
3.3. Cytogenetical damage
able 2 shows the cytogenetical damage induced byexposure to ECS, as indicated by the frequency ofmicronucleated NCE in peripheral blood. After 7days of exposure to ECS, animals belonging togroups (c)—(f), showed a four- to five-fold increasein the frequency of MN NCE, as compared to sham-exposed controls. All these differences were sta-tistically significant. After 14 and 28 days of expo-sure to ECS, the increase in the frequency of MNNCE was less striking but still statistically signifi-cant. Treatment with L-cystine/vitamin B6 alonedid not affect the frequency of MN NCE. Further-more, no significant difference could be observedamongst experimental groups of mice exposed toECS, either treated or untreated with L-cystine/vitamin B6.
Chemoprev
Table 2 Frequency of micronucleated (MN) normochro
nimals per group. Statistical analysis: *P < 0.05, **P < 0.01, and ***P < 0.001, as compared to controls.
194 F. D’Agostini et al.
Fig. 3 Histopathological appearance of the skin fromC57BL/6 mice either sham-exposed (A) or exposed whole-body for 6 months to ECS (B). Original magnification 40�.
lesions were variously localized on the mouse backand consisted of irregular areas of grey, hairlessskin. The mean (�S.E.) size of alopecia areas atthe end of the experiment, i.e., after 6 months ofexposure to ECS, was 1.65 � 0.21 cm2 in group (c)and 1.19 � 0.09 cm2 in group (f). This differencewas not statistically significant.
No mouse belonging to groups (a), (b), (d) and (e)was affected until the end of the experiment. Incontrast, the mice belonging to groups (c) and (f)underwent a time-dependent induction of alopecia(Fig. 2). In particular, after 14 weeks of exposure toECS, alopecia became evident in three out of eightanimals (38%) belonging to group (c) and in two outof eight mice (25%) belonging to group (f). After 22weeks of exposure to ECS, all animals belonging togroup (c) developed alopecia, whereas seven out ofeight mice (88%) belonging to group (f) wereaffected. After 24 weeks of exposure to ECS, allanimals belonging to group (f) had alopecia. Theincrease in the frequency of mice affected by alo-pecia became statistically significant in both groupsafter 16 weeks of exposure to ECS, as compared tosham-exposed controls. Differences between groups(c) and (f) were never statistically significant at anytime of the experiment.
3.5. Histopathology and apoptosis
Fig. 3 shows the microscopic appearance of repre-sentative samples of skin from a healthy area (A)obtained from a control (sham-exposed) mouse orfrom an area with alopecia (B) obtained from anECS-exposed mouse. The histopathological analysisof several areas of skin with alopecia revealed an
Fig. 2 Time-course induction of alopecia in ECS-exposedmice; either untreated [group (c), full circles] or treatedper os at the lowest L-cystine/vitamin B6 dosage (40/4 mg/kg b.w./day) [group (f), open circles]. No alopeciawas observed either in controls or in unexposed micetreated with the highest L-cystine/vitamin B6 dosage(1000/100 mg/kg b.w./day) or in ECS-exposed mice trea-ted with the medium (200/20 mg/kg b.w./day) or thehighest (1000/100 mg/kg b.w./day) dosage.
atrophy of epidermis, with a reduction in the num-ber of cell layers. Neither scarring nor any inflam-matory infiltrate in the dermis was detectable. Adecrease in the thickness of subcutaneous tissue wasalso observed. Hair follicles in the same areas werereduced in number and shortened. Rare and minutehair bulbs were detectable in alopecia areas. Mitoticactivity was very scarce or totally absent in thematrix of hair follicles (Fig. 4). In particular, themean (�S.E.) number of mitotic figures was2.5 � 0.38 in group (a), 2.1 � 0.35 in group (b),0.4 � 0.26 in group (c), 2.6 � 0.38 in group (d),2.4 � 0.38 in group (e) and 0.5 � 0.27 in group(f). The differences between groups (c) and (f)versus groups (a), (b), (d) and (e) were statisticallysignificant.
No appreciable difference in the histopathologi-cal aspect of alopecia areas could be observedbetween ECS-exposed mice and ECS-exposed micereceiving the lowest L-cystine/vitamin B6 dose (40/4 mg/kg b.w./day). No histopathological alterationcould be observed in healthy areas of skin obtainedeither from control mice or from ECS-exposed mice.
As assessed by analyzing 20 samples of healthyskin from mice belonging to groups (a), (b), (d) and(e), and 10 samples of alopecic skin from micebelonging to groups (c) and (f), apoptosis was well
Chemoprevention of smoke-induced alopecia in mice 195
Fig. 4 Mitotic activity in the matrix of hair follicles. Several mitotic figures (arrows) could be observed in healthy skinhair follicles obtained from control mice (A—C), whereas no mitoses could be detected in alopecic skin hair folliclesobtained from ECS-exposed mice (D—F). Original magnification 1000�.
196 F. D’Agostini et al.
evident in hair follicle cells at the edge of alopeciaareas. Apoptotic cells were distributed in all parts ofthe follicle.
4. Discussion
The present study showed the ability of ECS, asobtained by burning unfiltered research cigarettesin an automatic whole-body exposure system, toinduce alopecia in mice. We can therefore confirm,after our earlier experiment [6], that exposure toECS is associated with alopecia, and that the C57BL/6 mouse is a suitable model for investigating mod-ulation of ECS-induced alopecia by pharmacologicaland dietary agents. Variousmousemodels have beenproposed for the study of human hair loss [23]. Itshould be noted that, in all animal models, highdoses of toxicants administered for short periods oftime are needed to produce pathological effects,whereas humans are usually exposed to lower dosesfor longer periods of time. This circumstance ren-ders extrapolations to the human situation moredifficult, but in any case the findings obtained inanimal models, under well-defined and controlledexperimental conditions, are useful to define poten-tial health effects for humans and to assess theefficacy of putative protective agents.
The results of our analyses suggest that smoke-induced alopecia is associated with toxic, non-inflammatory and non-scaring disturbances of thehair cycle. In particular, ECS-related induction ofapoptosis in hair bulb cells nearby alopecia areasemerged as one of the most striking differencesbetween smoke-exposed and sham-exposed ani-mals. Apoptosis has been described as one of themain mechanisms involved in chemotherapy-induced alopecia in animal models [24—27]. There-fore, it can be assumed that apoptosis-blockingchemopreventive agents could exert a beneficialeffect against hair loss of toxic origin.
While a broad literature is available regardingmodulation of apoptosis by NAC in various cell types,either in vitro or in vivo [28], the ability of L-cystineto inhibit apoptosis has been so far demonstratedonly in vitro [29—32]. On the contrary, this activityhad never been investigated earlier in vivo. Thisproperty is biologically plausible, since a severecystine deficiency can have marked effects on thecellular redox state [33]. Redox mechanisms controlgene expression, cell proliferation and apoptosis. L-Cysteine and methionine are the only amino acids inproteins that undergo reversible oxidation/reduc-tion under biological conditions and, for this reason,the cysteine/cystine couple is an important node inthe circuitry for redox signaling and control [34]. In
addition, enteral or parenteral L-cystine is amongsteffective precursors of cysteine for tissue GSHsynthesis [35]. GSH plays very important roles inantioxidant defense and regulation of cellularevents, including cell proliferation and apoptosis.
Our study demonstrated that the L-cystine/vita-min B6mixture does neither prevent the loss of bodyweight produced by ECS in mice nor protect ECS-exposed mice from a systemic genotoxic damage, asevaluated by assessing the frequency of micronucleiin peripheral blood erythrocytes. In a previous study[36], GSH and its oxidized form (GSSG), L-cysteineand L-cystine failed to inhibit the induction of sisterchromatid exchanges in cultured hamster cellsexposed to nitrogen oxides. On the other hand, L-cystine has been demonstrated to protect miceagainst the toxicity induced by paraquat, a pesticideexerting genotoxic effects in various organs due toproduction of reactive oxygen species (ROS) [18,19].CS produces large amounts of ROS [37] and gener-ates an increased burden of peroxynitrite, whichcauses apoptosis [38]. Therefore, it could behypothesized that the protective effects exertedby oral administration of L-cystine against ECS-induced alopecia, as demonstrated in the presentstudy, should be mainly ascribed to a variety ofmechanism which inhibit apoptosis. Presumably, L-cystine does not possess direct anti-apoptotic prop-erties but, like NAC and other chemopreventiveagents, it works upstream by counteracting apop-tosis-triggering events [28].
While no information is available in the literatureabout the apoptosis-modulating properties of vita-min B6, it has been demonstrated that deprivationof individual B-group vitamins in the culture mediaincreases cell death rate, mainly via apoptosis, inhybridoma cell cultures [39]. An in vivo studyshowed that mice receiving a vitamin B6-depleteddiet developed abnormal apoptosis in the thymus[40]. Oral administration of vitamin B6 resulted in aprotective effect against chromium-induced oxida-tive stress in rat liver [41]. In addition, this vitaminhas been demonstrated to possess antimutagenic[42] and anticarcinogenic [43,44] properties. There-fore, it is arguable that the addition of vitamin B6contributed to the chemopreventive action of L-cystine.
The chemopreventive effect of the L-cystine/vitamin B6 mixture on alopecia, as observed inthe present study, was dose-dependent. It was fullyobtained at the two highest dosages tested, whereasno effect could be observed at the lowest dosage.The threshold of efficiency of the L-cystine/vitaminB6 mixture lies in a very narrow dosage zone,between 40 and 200 mg/kg b.w. for L-cystine and4 and 20 mg/kg b.w. for vitamin B6. Presumably, at
Chemoprevention of smoke-induced alopecia in mice 197
the lowest dosage an insufficient plasmatic concen-tration was reached. On the other hand, no toxic orside effects were observed after oral administrationfor 6 months at all dosages tested, and this is inaccordance with the fact that toxicity of L-cystineand vitamin B6 is low [45—47].
In conclusion, from the results of thepresent studyit can be stated that oral administration of a L-cystine/vitamin B6 mixture with the diet is an effec-tive chemopreventive treatment against smoke-induced alopecia inmice. Further studies are neededin order to evaluate the contribution of the individualadministration of L-cystine and vitamin B6 to thisprotective effect. Taking into account that all resultsobtained in animal models cannot be automaticallyextrapolated to humans, further studies are neededbefore suggesting this chemopreventive protocol tosmokers, either active or passive. In fact, it will benecessary to test their efficacy against smoke-induced alopecia in clinical trials in humans. Further-more, it should be stressed that the better way ofpreventing smoke-inducedalopecia inactive smokersis cessation of smoking which, without any doubt,results in the prevention of several other deadlychronic degenerative diseases.
Acknowledgement
This study was supported by grants from the Uni-versity of Genoa.
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