Paper in Press, No 2013_497

Cancer chemotherapy is associated with serious side effects, including temporary hair loss and impairment of pigmentation. We suspect that ectopic melanin deposition occurring due to chemotherapy may add to these effects worsening the already unpleasant symptoms. We associated the ectopic occurrence of follicular melanin after chemotherapy with splenic melanosis - an interesting example of extradermal melanin localization - and we expected an increase in splenic melanin deposition after chemotherapy. Using the C57BL/6 murine model of synchronized hair cycle induced by depilation, we visualized splenic melanin by means of several histological and histochemical protocols of staining: hematoxylin and eosin, May-Grünwald-Giemsa and Fontana-Masson. Unexpectedly, the splenic deposition of melanin decreased due to application of cyclophosphamide (i.p. 120 mg/ kg body weight on day 9 post depilation). The drop was abrupt and lasted for at least 5 days (day 13-18 post depilation), as compared with normal hair cycle. Moreover, in mice with normal, depilation-induced hair cycle we observed a similar drop shortly before entering catagen (day 15 post depilation), followed by a slow and partial increase in splenic melanization up to day 27 post depilation in both groups. We conclude that cyclophosphamide negatively affects splenic melanization and/or extradermal transfer of ectopic melanin from the dystrophic hair follicles, but the most powerful down-regulator of splenic melanosis is normal and dystrophic catagen - the phase of hair follicle involution and re-modelling.


INTRODUCTION
One of the most acute side effects of cancer chemotherapy is hair loss and impairment of hair pigmentation.This phenomenon adds to the overall depressive condition of patients making them feel devastated and is sometimes a reason for giving up chemotherapy (McGarvey et al., 2001).Understanding the underlying mechanisms is, consequently, an important factor of the success in cancer therapy.One of the related problems is the fate of epidermal and follicular melanin which is initially deposited ectopically in response to chemotherapy (Braun-Falco, 1961).This melanin is toxic in itself (Slominski et al., 1996;Swartz et al., 2005;Wood et al., 2009) and affects the general condition of the organism.
Moreover, the pigmented tumors (melanotic melanoma) may also reveal symptoms of deregulated melanogenesis, which may influence the process of therapy (Plonka et al., 2003;Lazova et al., 2010).This makes the topic of extradermal melanin transfer important also from the oncological point of view (Michalczyk et al., 2009).
Melanin, an amorphous polymer responsible for skin and hair pigmentation, may be also found in other organs.In higher vertebrates, including humans (Wasserman, 1967), melanin has been reported, besides skin, in visceral organs, and its presence and origin in such extradermal locations is enigmatic.Pigmentation of murine spleens was noted for the first time by Weissman (1967).Early studies delivered numerous hypotheses concerning the identity of the pigment.In (1978) Crichton et al. identified the pigment as lipofuscin.In (1989) Veninga et al. postulated deposition of hemosiderin as the cause of spleen pigmentation.Work of Sundberg et al. (1991) and van der Heijden et al. (1995) finally confirmed the presence of melanin.Simultaneously, they excluded lipofuscin -the pigment characteristic for ageing, since the phenomenon was observed in young animals.The presence of melanin was directly proved by Plonka et al. (2005) by means of electron paramagnetic resonance (EPR, also called electron spin resonance, ESR).In (2009) Michalczyk et al. indicated that partial melanization of spleens is observed in young C57BL/6 mice (younger than 10 weeks) with synchronized hair cycle.Meanwhile, over one-year-old mice revealed no splenic melanosis, instead, 'melanin debris' could be observed in some of these old mice.
Excessive melanin deposition (melanosis) of inner organs is a long-studied phenomenon (Wasserman, 1967).It has recently turned out that melanopho/res play an important part in amphibian metamorphosis, which includes total re-building of the tissues, including skin.An interesting manifestation of this process is deposition of * e-mail: przemyslaw.plonka@uj.edu.pl*Presented at 40th Jubilee Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University "Contemporary insights into cancer.Risk, perspectives, expectations", February 16-21, 2013 U n c o r r e c t e d P a p e r i n P r e s s melanin in the liver and other visceral organs of the animals (Divya et al., 2010).Understanding the origin and mechanism of splenic melanization in mice is, therefore, important from the point of view of general biology, evolution of the skin, and experimental dermatology (Rakers et al., 2010).The most important, hypothetical extrasplenic source of the murine spleen melanin are the hair follicle melanocytes.
The mammalian hair follicle is a mini organ, which undergoes continuous remodelling through the whole animal life in the process called hair cycle (Chase, 1954;Müller-Röver et al., 2001).In many animals, including mice, it is synchronized over big areas of skin (Dry, 1926).The hair grows and gains melanin only in anagen -the stage of hair growth.In the subsequent stagecatagen (very well synchronized in young C57BL/6 mice, and followed by telogen, the "resting" stage), follicular melanocytes undergo massive apoptosis, and the melanin-containing apoptotic bodies are phagocytosed initially by the Langerhans cells (Tobin, 1998) and then transferred further, probably as far as to the spleen.In the meantime melanin undergoes partial degradation (Borovansky & Elleder, 2003;Plonka et al., 2005).Therefore, splenic melanization must be correlated with the progress of the hair cycle.
Impairment of the hair cycle and the related melanin production is often associated with ectopic deposition of the pigment in the hair follicle outside the hair shaft.Such pathological melanization is a side-effect of cancer chemotherapy with cyclophosphamide (CYP) in humans (Braun-Falco, 1961), and in model mice (Kostanecki et al., 1967).This is a rationale to suppose that in such a case a particular increase in splenic melanin deposition can be expected.On the other hand, in normal mice we found less splenic melanin in early telogen than in late telogen (Plonka et al., 2005) even if one of the ways through which hair follicles can recover from CYP-related dystrophy is to enter dystrophic catagen followed by dystrophic telogen (Paus et al., 1994a).It creates the necessity to assess splenic melanosis over the whole hair cycle, in its every stage, with and without CYP administration.The presence of melanocytes in murine skin is limited almost exclusively to hair follicles (Chase, 1954;Slominski et al., 2005).Since those laboratory rodents exhibit the wave-like type of hair growth (Dry, 1926;Chase, 1954;Chase & Eaton, 1959), synchronization of every stage of the hair cycle is possible, which is even more prominent in the case of the depilation-induced cycle (Paus et al., 1990).Therefore, this model is of particular suitability to study correlation between splenic melanosis and hair cycling.
One of the most popular and widely applied is hematoxylin and eosin staining (Mayer, 1891;1904).It employs two dyes specific for different cell compartments.Hematoxylin, a blue dye, possesses affinity to basophilic (acidic) structures within the cell (mainly chromatin, therefore the nucleus is visualized very efficiently).To obtain a sharp contrast with the blue hematoxylin signal, the staining with purple eosin Y is used.This dye shows affinity to acidophilic structures (usually positively charged, because eosin is an acid).In practice, eosin stains the entire cytoplasm and cell membrane of various types of cells (Romeis, 1991;Avwioro, 2011).
Another staining method whose specificity is based on electrostatic interactions between the dye and the target structures is May-Grünwald-Giemsa stain (MGG).May-Grünwald staining solution is composed of eosin Y and methylene blue.Giemsa solution is applied separately and is composed of azure-type dyes: azure II is a mixture of methylene blue and its derivative in 1 : 1 proportions, and of eosin.The Properties of azures and methylene blue are similar to those of hematoxylin -these dyes, carrying a positive charge, stain nuclei and basophilic cells (Barcia, 2007).
The third staining procedure is based on the reduction of diamminesilver(I) nitrate to metallic silver(0) under the influence of reductive agents present in the cell.This method, described by Fontana (1912) and Masson (1914), was applied by Fontana for visualization of a spirochete (Treponema pallidum), and in its basic version it is known as Fontana-Masson (FM) staining.Masson applied 'silver solution' for visualization of neuronal structures (Moore et al., 2001).Cells which reduce diamminesilver(I) nitrate to metallic silver(0) are referred to as argentaffins.A positive result of such 'silver stain' is mostly correlated with the presence of serotonin (Barter & Pearse, 1955) and other biogenic amines such as dopamine or ephedrine (Lundqvist et al., 1990).Melanins are composed mainly of derivatives of dihydroxyindole monomers that can be oxidized by diamminesilver(I) nitrate and thus melanins can be marked with metallic silver(0) (reaction 1).EPR spectroscopy is widely used to investigate melanin-containing tissues.Paramagnetic properties of this pigment were pointed out for the first time in 1954 (Commoner et al., 1954).Since then EPR has been applied to quantitative (Pilas & Sarna, 1985;Slominski et al., 1994), and qualitative (Sealy et al., 1982) assays of melanin and of its microenvironment in the tissue (Felix et al., 1978;Plonka et al., 2005).It is, however, not able to localize melanin spatially in the spleen tissue instead one is bound to use standard histological methods.
In the present paper we compared HE and MGG staining and the FM method, which is melanin-specific in terms of quality and potential to visualize melanin and the contours of individual cells, in murine C57BL/6 spleens.The presence of melanin was verified independently by means of EPR spectroscopy.We checked the presence of splenic melanosis on subsequent days after induction of the hair cycle by depilation.We also examined whether chemotherapy with CYP increases or decreases splenic pigmentation, and discussed what implications it may have for cancer treatment.

MATERIALS AND METHODS
Instruments.Paraffin slices were prepared using a manual Finesse 325 microtome (Thermo Shandon, Runcorn, UK), images of stained tissues were taken by a reversed Eclipse Ti microscope (Nikon Corporation, Tokyo, Japan) equipped with the Nis elements F 3.0 imaging software (Nikon Corporation, Tokyo, Japan), and an analog camera (PENTAX ME, Asahi Opt.Co., Tokyo, Japan) equipped with 1:4.5/8-20 mm Soligor MC lenses (Sun Optical Co., Ltd., Ichikawa, Japan) adjusted for macrophotography.EPR spectra were recorded by an E-3 Biological material.The biological material was collected over a long time and during several different experiments, all of which were approved by the 1 st Local Committee for Animal Research in Kraków (221/95, 303/97, 15/OP/2004).Female, 6-8-week-old C57BL/6 mice (Animal Breeding Facility, Silesian Medical Academy, Katowice-Ligota, Poland) were selected for depilation based on the pink color of their back skin (all hair follicles in telogen, Paus et al., 1990).Depilation was executed in ketamine anesthesia by application of melted 1:1 mixture of beeswax and gum rosin and peeling out the hair coat after hardening, according to Paus et al., (1990).On day 9 post depilation (p.d.) the CYP-treated animals were administered a single i.p. dose (120 mg/kg body weight) in a small volume (ca.0.1-0.2ml) of saline (Paus et al., 1994a;Hendrix et al., 2005), while the control animals were given vehicle.At subsequent time points (see Fig. 1) the animals were shaved if necessary, photographed, and killed by cervical dislocation in deep ketamine anesthesia, whereupon the skin was separated at the level of subcutis, spread on a piece of cardboard, and fixed in buffered 5% formalin (phosphate buffer, pH = 7.4).We determined the stage of the hair cycle and dystrophy of the hair follicles based on the tabularized criteria of histomorphometry (Müller-Röver et al., 2001;Hendrix et al., 2005).
The spleens of animals were carefully examined on necropsy for macroscopic evidence of melanosis and fixed in the formalin solution for histology.We estimated the area of melanosis and calculated the percentage of melanotic spleens per a given experimental group of mice.A part of spleens was frozen in liquid nitrogen for EPR measurement.
Preparation of tissue slices for histology.To prepare paraffin blocks, the spleens, after a long fixation in formalin, were rinsed with water (24 h), dehydrated in the series of aqueous solutions with increasing concentration of ethanol (50%-1 h, 70%-2 h, 80%-2h, 96%-12 h, 100%-40 min, 100%-1 h) and then in methyl salicylate (4 h), xylene (10 min) (all at room temperature) and embedded in paraffin blocks.Shortly before cutting into 5 μm slices the paraffin blocks with embedded spleens were additionally cooled in icy water.We found it an important step preventing the spleen tissue from crumbling.
Pre-treatment of slides with tissue sections.Paraffin sections of spleen tissues on Polysine ® slides were deparaffinized by: a) incubation of the slides in 56°C for 20 min to melt paraffint and to attach the tissue to the surface of glass, b) dissolution of the melted paraffin in xylene (5 min) and then immersing the slides in fresh xylene several times, and c) re-hydration of the tissue by immersing the slides in a series of aqueous solutions with decreasing concentration of ethanol (100, 96, 80, 70, 50, 0% of ethanol, v/v, respectively).
Hematoxylin and eosin (HE) staining.The deparaffinized slices were incubated in hematoxylin working solution (12 min) at room temperature; flushed with tap water (15 min), counterstained for 1.5 min in 0.1% ethanol solution of eosin acetified with a few drops of acetic acid; dehydrated in the ethanol series (70%, 2 × 96%, 2 × 100%, v/v, respectively), 2 × xylene (all at room temperature), sealed in balsam and covered (Romeis, 1991).May-Grünwald-Giemsa (MGG) staining.Working solution of Giemsa was prepared by mixing 0.5 ml of concentrated Giemsa stock solution with 200 ml of distilled water.The MG working solution was prepared by mixing 15 ml of MG stock solution with 160 ml of distilled water.Deparaffinized slides with samples were incubated in MG working solution for 20 minutes at 37°C in a Coplin jar covered with aluminium foil to protect against light.The samples were then introduced into Giemsa working solution for 40 minutes at 37°C, immersed quickly in 0.15% solution of acetic acid to obtain neutral pH, washed in distilled water, dehydrated, sealed  in balsam and covered with glass coverslip like for HE staining (Zawistowski, 1983).
Fontana-Masson (FM) staining for melanin.Due to the sensitivity of the applied ammoniacal silver nitrate, it was prepared immediately before the staining procedure.Concentrated ammonia was added drop by drop to 20 ml of 10% solution of silver nitrate until the white precipitate of silver(I) oxide was almost dissolved (hardly noticeable opalescence).The obtained solution was diluted by adding 20 ml of distilled water, then filtered and stored at 4°C in a bottle protected from light.
Deparaffinized slides with samples were rinsed in distilled water and incubated in silver solution for 40 minutes at 56°C in a Coplin jar covered with aluminium foil, while the control slides were incubated similarly in distilled water.After washing in distilled water, the slides were incubated in 5% sodium thiosulphate (3 min), flushed with tap water (3 min), counterstained in 0.5% aqueous solution of toluylene red (neutral red) for 5 min to stain nuclei, flushed with tap water again, dehydrated by immersion for a few seconds in the ethanol series (70, 96 and 100%, v/v; × 2 each) and then in xylene (10 min), whereupon the slides were conserved with balm and covered with a glass coverslip (Bancroft & Stevens, 1982).
EPR measurements.Whole amelanotic and melanotic spleens were analyzed in a Dewar quartz tube in liquid nitrogen (77 K) at X-band (ca.9.2 GHz), 4 mW microwave power and 5Gs modulation amplitude.Amplification of the signal was set at 8 × 10 5 (melanotic spleens) or 1.25 × 10 6 (amelanotic spleens).Each spectrum was averaged from 3 scans at 200 s scan time and 0.3 s time constant.
Statistical evaluation of the data.We examined 1-5 experimental groups, each of 2-8 animals, per time point (5-22 animals per time point).We expressed the average melanotic area of a spleen (% of the total area) as the mean of the pooled data for a time point ± S.E.We also calculated the total percentage of spleens revealing melanosis per time point (i.e.pooled groups examined at a given time point).Altogether we examined 161spleens (92 control and 69 CYP-treated).The two-tailed independent Student's t-test was used to evaluate the statistical significance of the differences between the means, and the Snedecor F test to assess the significance of the differences in variances.The differences were accepted as significant for P < 0.05.

Visualisation of splenic melanin
Splenic melanosis manifests itself macroscopically (Fig. 2A-D) and has been described in C57BL mice as "black spots" (Weissman, 1967).It is a yes-or-no phenomenon, and for yet unknown reasons in all investigated groups there are animals with either melanotic, or non-melanotic spleens.As a result, in a given animal the existence of splenic melanosis cannot be determined without laparotomy (Weissman, 1967; van der Heijden et  , 1995;Plonka et al., 2005).On the other hand, as the black spot is sharply separated from the amelanotic background, it is easy to assess melanosis macroscopically as the average percentage of the melanotic area of spleens and the percentage of mice revealing melanosis in a given group.
We have shown here that melanin granules can be histologically visualized by means of all the applied methods.As it can be seen in Fig. 2E-P, the depicted melanin granules are dyed in a comparable way -the gamut of colors ranges from yellow-brown to black.On the basis of the properties of the applied chromophore molecules, one may attribute the positive result of staining to basic dyes (methylene blue and its derivatives) due to the negative net charge of melanin.However, a touch of yellow (e.g.Fig. 2F) suggests a contribution of eosine Y, possibly thanks to the π-π stacking interaction (similar to that present in the aggregates of eumelanin promolecules; Meredith & Sarna, 2006).
Metallic silver (Fig. 2M-P) has been shown to be an effective marker of melanin granules in Fontana-Masson staining (Slominski et al., 1994;1996;2004;2005).However, to prevent 'false positive' results, the time of incubation in the solution of diamminesilver(I) nitrate should not be exceeded.According to our observations, a significant concentration of melanin may sometimes lead to the excessive intensity of silver deposition, and prolongation of the incubation time may lead to non-specific precipitation of silver(0), possibly due to other mild reducing agents apparently present in cells.Nevertheless, while the time of incubation in 'silver solution' should always be carefully considered, sometimes a slight prolongation or even moderate heating of the bath increases the quality of the picture.To sum up, a negative control (incubation without silver nitrate) is always indispensable.
It should be stressed that neither of the two 'nonsilver' methods (HE or MGG), stains melanin specifically.Their role is to increase the contrast between the background and melanin granules.On the contrary, FM staining is the method which directly, histochemically stains melanin and as such, it may be applied to confirm the reducing potential of the previously visualized granules.
A perfect, independent method to determine directly the presence of melanin in a given sample is EPR spectroscopy (Sarna & Plonka, 2005).The results of EPR assay of the spleens of young mice with the presence (mel+) and the absence of visible melanization (mel-) are shown in Fig. 3. Additionally, an EPR spectrum of DPPH was recorded as a free radical standard (g = 2.0037 ± 0.0002; Wertz & Bolton, 1984).The shape of the splenic sig-  U n c o r r e c t e d P a p e r i n P r e s s nal and its position prove that all the observed radical centres are isolated and each contains only one unpaired electron not coupled with any other electron or nuclear spin, therefore confirming the presence of eumelanin in the spleens (Meredith & Sarna, 2006).
The mel-signal is distinctly weaker than both the mel+ ones, and originates from endogenous free radicals associated with mitochondrial respiration (Fig. 3; Slominski et al., 1994;Plonka et al., 2003;Elas et al., 2008).Since, due to a relatively small amount of melanin in the spleen, the melanin-derived signal is rather weak as compared to the non-melanin one, the quantitative EPR determination of melanin may be charged with a relatively substantial experimental error.Moreover, despite direct and unambiguous correlation between the presence of melanin spots on the tested spleens and the occurrence of the EPR signal characteristic of eumelanins, EPR spectroscopy does not provide resolution on the 'cellular' level.The recently reported attempts to employ the technique of EPR imaging (EPRI; Godechal et al., 2012) did not provide a satisfactory resolution.As such, EPR constitutes a complementary approach to histological staining protocols which gives an insight into the distribution of melanin in the tissue.

Hair cycle and melanogenesis
In the murine trunk skin mature, melanin-producing melanocytes are present only in the anagen hair follicles (Slominski & Paus, 1993;Slominski et al., 1994).Macroscopically it is manifested by a grey-to-black color of the skin (Fig. 4B, C), in contrast to the pink telogen skin (Fig. 4A, I), totally devoid of mature (i.e.melaninproducing) melanocytes (Fig. 5A).The changes of macroscopic skin are particularly well noticeable in the depilated skin (Fig. 4A, B).The active melanogenesis starts about day 5 p.d. (anagen III), and lasts to the end of anagen VI (day 15-16 p.d.; Müller-Röver et al., 2001).The hair follicle in anagen VI is fully developed and contains a strongly melanized hair shaft and a developed population of mature, melanin-producing melanocytes (Fig. 5B,  C).
If a strong alkylating chemotherapeuticum, like CYP, is applied on day 9 p.d., i.e. at the beginning of anagen VI (Fig. 1, 4B, 5B), the hair follicles react with acute dystrophy (Braun-Falco, 1961;Kostanecki et al., 1967;Paus et al., 1994a;Slominski et al., 1996).The whole population of multiplying melanocytes and keratinocytes undergo abrupt apoptosis (Hendrix et al., 2005), resulting in alopecia (Fig. 4D, F, H), whereas in the control skin (Fig. 4C,  E, G) the anagen VI hair follicles still produce normally pigmented hair shafts (Fig. 5B, C).As a result, on day 15 the CYP-treated animals are almost completely devoid of hair (Fig. 4H).The color of the skin, however, still indicates the presence of melanin.And indeed, ectopic, extracellular melanin deposition (Fig. 5D-F) is associated with both possible pathways that allow the dystrophic hair follicles to recover from the CYP-induced impairment: via dystrophic anagen (Fig. 1, 5D), or via dystrophic catagen (Fig. 1, 5E) and telogen (Fig. 1, 5F), each accompanied by a transient hair loss (Paus et al., 1994a;Slominski et al., 1996;Hendrix et al., 2005).This may be particularly well appreciated in comparison to normal anagen VI hair follicles, in the histological pictures with the FM staining of melanin (Fig. 5C), accompanied by HE visualization (Fig. 5B).Actually, the CYP-induced dystrophy may be very strong (Hendrix et al., 2005) and it affects the apparatus of hair follicle melanogenesis, causing ectopic (hair root sheaths, sebaceous gland etc.) melanin deposi-tion and clumping of the amorphous melanin mass in the hair channel (Slominski et al., 1996;Hendrix et al., 2005;Paus et al., 2013).We suspected this ectopic melanin (Fig. 5D-F) to be the source of the presumptive increase in melanin deposition expected by us to occur in the spleen after CYP-induced alopecia.

Splenic melanosis during normal and CYP-impaired hair cycle
In Fig. 6 we have shown the summary of our extensive observations of the correlation between splenic melanosis and the progress of hair cycle, at particular time points after depilation.The samples were collected over a long time and cumulated from several independent experiments and from the control groups of other experiments.As the splenic melanization is a "yes-or-no" phenomenon (Plonka et al., 2005), only such an extensive study could deliver any quantitative comparisons.
And indeed, as it can be seen in Fig. 6A, the intensity of splenic melanosis changes according to the progress of normal hair cycling.The most important observation concerns the period shortly preceding and following the onset of catagen, with a strong drop in the melanization (Fig. 6A, B, day 15).This drop was followed by an increase during the subsequent long telogen period, confirming our earlier observations (Plonka et al., 2005).Catagen, as other stages of the synchronized hair cycle, is a phenomenon systemically influencing the murine organism (Slominski et al., 1997;Paus et al., 1999).Here we show its association with a drop in splenic melanosis in C57BL/6 mice.
If this is the case, the drop should be observed also in the CYP-treated animals, moreover, earlier than in the controls.And indeed, we found it happening, as shown in Fig. 6 (day 13).The decrease is abrupt and followed by a gradual re-storage of melanin in the spleens, up to the level of the control (Fig. 6, days 13-27).This can be confirmed with macroscopic (Fig. 2C, D), and histological examination of the spleens, revealing smaller and fewer deposits of melanin in the organ using the FM staining (Fig. 2P) confirmed by the MGG (Fig. 2L), and HE (Fig. 2H) visualization.
As chemotherapy with CYP influences strongly the follicular melanogenesis, we expected a reverse effect in the spleens.We expected scavenging of the abundant CYP-induced ectopic melanin mass (Fig. 4D-F) by melanophages, and consequently, its massive and delayed deposition in the spleen.Meanwhile, we have found poorer melanization of CYP-treated than untreated spleens for at least 6 days (Fig. 2D, P, L, H, 6 day 13-18) and a slow re-melanization of the spleens to the level of the control.
The overall picture of the splenic melanosis in parallel to the progress of normal or CYP-disrupted hair cycle is as follows.There are three clear phases of the phenomenon.Initially, melanosis is quite high, and culminates on day 9. Next, it drops down till day15, i.e. the day directly preceding catagen.Finally, it increases slowly back on the border of statistical significance for the next 12 days (during telogen).If CYP is administered on day 9, the drop is much more abrupt, (the minimal value on day 13), followed by a 15-day re-pigmentation .
However, on day 18 the CYP-treated spleen pigmentation is still significantly lower than the control.The CYP-treated spleens reveal decreased pigmentation longer than the normal ones.It can be manifested both macroscopically (Fig. 2C, D), and histologically (Fig. 2G, H, K, L, O, P), as well as supported quantitatively (Fig. 6).
The peak of splenic melanization can be observed on day 9, i.e. at the onset of anagen VI.As this is also the point of the highest activity of the hair-cycleassociated melanin synthesis (Plonka et al., 1995), splenic melanosis could be associated with skin melanogenesis, if it was not for the presence of splenic melanosis in the absence of melanin synthesis in the telogen skin (from day 18 to day 27; see Fig. 4I ).During this time the CYP-treated skin is a mosaic of all the phases of normal and dystrophic hair cycle (Plonka et al., 2006; see also Fig. 4J).The lowest intensity of splenic melanization is associated with the onset of dystrophic anagen and catagen (Fig. 5D-F), which, after injection of CYP on day 9, can be observed on day 12-13, i.e. earlier than in the control animals (Paus et al., 1994a;Hendrix et al., 2005).Concluding, our observations strongly suggest that the primary factor responsible for splenic melanosis is the stage of the hair cycle, not the content of ortho-or ectopic melanin in the hair follicle.However, since even in the CYP-treated mice, ca.one fifth of the spleens (Fig. 6B) revealed weak (Fig. 6A) melanosis on day 13 we still cannot explain the reason of such a "digital" ("zero-or-one") manifestation of this phenomenon in C57BL/6 mice.

CONCLUSIONS
The results of our experiments lead to important practical implications.First of all, CYP abruptly and for a considerable period decreases splenic melanosis despite deposition of ectopic melanin in the CYP-treated skin.During normal catagen melanin is released from the apoptotic melanocytes and keratinocytes in the form of apoptotic bodies easy to phagocyte and partially degrade (Tobin, 1998).CYP causes massive apoptosis of various cells and deposition of melanin in the form of amorphous agglomerates, which are probably difficult to be absorbed (Paus et al., 1994a;Hendrix et al., 2005).Moreover, the population of phagocytic cells (Meinhardt et al., 2011;Hirsch et. al., 2004;Quéméneur et al., 2003) including splenocytes (Teryukova et al., 2011) may be transiently decreased and their capacity of devouring melanin -transiently limited.This means that the "naked", toxic melanin remains in the CYP-treated skin (see Fig. 5D-F), where it may add to the bad condition of skin and, consequently, of the whole organism (Chuong et al., 2002;Tobin, 2006).CYP may also negatively influence the spleen itself, which, besides poorer melanosis, may be manifested by e.g. a smaller size of the organ (Fig. 2C, D; Teryukova et al., 2011).
The second important conclusion concerns the systemic effects of the hair cycle: catagen, both normal and dystrophic, is always associated with a drop in the splenic melanosis.Whatever mechanism stands behind this phenomenon, catagen in the depilation-induced hair cycle causes systemic effects.Therefore, regulation of this phase is probably dependent on factors affecting the whole organism.
U n c o r r e c t e d P a p e r i n P r e s s n{[Ag(I) (NH3)2] + (aq) + e -→ Ag (0) ↓ + 2NH 3 (aq)} melanin(reduced) → melanin(oxidized) + ne -Reaction 1.Schematic illustration of the principle of silver(I)-dependent oxidation of melanin.

Figure 1 .
Figure 1.Experimental design.Empty circles, harvesting of spleens; black circles, harvesting of skin.Below, general scheme of normal hair cycle in C57BL/6 mice, and dystrophic hair cycle after administration of cyclophosphamide (i.p. 120 mg/kg body weight) on day 9 post depilation (according to Müller-Röver et al., 2001, and Hendrix et al., 2005).

Figure 4 .
Figure 4. Macroscopic manifestation of hair cycle and cyclophosphamide-induced alopecia in C57BL/6 mice.(A) female, 7 week-old mice were depilated in telogen (day 0, note the pink color of the back skin), and (B) injected i.p.CYP (120 mg/ kg body weight ) on day 9, i.e. at the beginning of anagen VI (note the dark-blue color of the skin due to intensive melanogenesis).The first symptoms of alopecia were noticeable on day 13 p.d. (D -white arrows), and progressed through day 14 (F) up to day 15 (H), while in control mice the hair follicles produced a normally pigmented hair coat (C, E, G -vehicle control, day 13, 14, 15 p.d., respectively).Note the deep-blue color of the bald skin on day 15 p.d. (H).The hair coat of the CYP-treated animals was still recovering on day 27 (J) when the skin still revealed symptoms of dystrophy (impaired pigmentation -d) beside regions of skin with hair follicles in the 2nd (recovery) anagen (r), while the hair of control mice (I) had been for a long time in deep telogen (note the pink color of the skin uncovered by shaving).

Figure 5 .
Figure 5. Histological manifestation of hair cycle-related melanogenesis and its impairment due to cyclophosphamide in C57BL/6 mice.(A) a telogen hair follicle without melanocytes on day 0. (B, C) normal anagen VI hair follicles with normally pigmented hair shafts (blue arrows) on day 9 (B), and 15 p.d. (C).(D, E, F) dystrophic hair follicles on day 15 p.d in late dystrophic anagen (white arrows), late dystrophic catagen (black arrows) and dystrophic telogen (red arrows).The criteria of dystrophy -according toHendrix et al., (2005).The black and white arrows point at both the granules of ectopic melanin, and melanin clumping in the hair channel (red arrows).This melanin was expected to be scavenged by melanophages and transported to the spleen.A, E, F -HE; B, C, D -FM.Scale bars, 100 µm.

Figure 6 .
Figure 6.Splenic melanosis at particular time points of depilation induced, normal (CTRL) and dystrophic (CYP) hair cycle in C57BL/6 mice.(A) average area of melanosis (means ± S.E.M.); (B) total percentage of melanotic spleens observed at a particular time point.CYP, cyclophosphamide (i.p. 120 mg/kg body weight on day 9 p.d.).*P < 0.05, **0.05 > P ≥ 0.01.U n c o r r e c t e d P a p e r i n P r e s s