Introduction

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It is known that drugs associate with synthetic melanin and melanin that is obtained from cuttlefish (Sepia officinalis) in vitro (Shimada et al., 1976; Potsch et al., 1997). Additional studies have found differences in drug concentrations among differently pigmented hair (Shimada et al., 1976; Gygi et al., 1995; Cone and Joseph, 1996; Joseph et al., 1997; Potsch et al., 1997; Rothe et al., 1997; Knorle et al., 1998; Slawson et al., 1998). Emmerich et al. (1998) found that cultured melanocytes accumulated more cocaine than did keratinocytes. We have demonstrated pigment-dependent differences in deposition of radiolabeled serum constituents in vivo (Stout et al., 1998a). The role of pigment in the deposition of drugs into hair has raised questions of the potential for increased sensitivity in people with greater pigment concentrations (Cone and Joseph, 1996) and of a potential for ethnic bias in hair testing.

To determine the subcellular location of drugs deposited in developing hair, we administered three drugs of forensic importance. [³H]Cocaine, [³H]flunitrazepam, and [³H]nicotine were systemically administered to pigmented and nonpigmented animals. The distribution of these compounds into the hair was examined by microautoradiography of skin sections that contained developing hairs (Stout and Ruth, 1999). We found that all three drugs were deposited primarily within the melanosomes and melanocytes of the hair follicle and that this association was evident within 5 min of dosing.

From our previous work with radiolabeled serum constituents (Stout et al., 1998a), we found that the concentration of ⁴⁵Ca²⁺ in pigmented hair was dramatically higher than that found in nonpigmented hair. We also found that [¹⁴C]urea and [³⁵S]cysteine were deposited in both pigmented and nonpigmented hair in much the same concentrations. In this study, we report the histologic pattern of deposition of the serum constituents in the developing hair.

	Materials and Methods

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Mice (36 C57 and 36 BALB/c) were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained under approved protocols at the University of Colorado Health Sciences Center. Mice were obtained at 23 days of age as have been used in previous studies (Stout et al., 1998a,b; Stout and Ruth, 1998, 1999). Mice undergo a period of synchronized hair growth (anagen), which begins at 23 days of age (Green, 1966). This assured that the hair was growing, and thus actively incorporating compounds, during the dosing period. Because BALB/c mouse hair does not contain any pigment and C57 mouse hair contains almost exclusively eumelanin (Hamilton et al., 1974) we were able to assess the effects of pigment on deposition.

Radioisotopes were obtained from NEN (Boston, MA). ³⁶Cl was obtained as H[³⁶Cl] (13.4 mCi/g) and was prepared in 100 mM, pH 6, phosphate buffer for injection. It is unlikely that this had any adverse effect on the compound. ⁴⁵Ca²⁺ was obtained as [⁴⁵Ca]Cl₂ (22.6 mCi/mg) and was dissolved in 5% glucose solution for injection. [¹⁴C]urea (57.4 mCi/mmol) and L-[³⁵S]cysteine (1075 Ci/mmol) were also prepared for injection in 5% glucose solution for injection.

For each tracer series, three mice for each time point were administered a single i.p. dose (100 µl containing approximately 1 µCi) of the tracer solution. Mice were sacrificed by asphyxiation in CO₂ at 5 min, 6 h, or 24 h after dosing. Skin was immediately harvested from the scapular region of each mouse.

The method used for the preparation, exposure, development, and microscopy of the skin sections is described in Stout and Ruth (1999). However, due to the higher energy of emission by the tracers used in this study, Kodak NTB3 autoradiographic emulsion was used.

In brief, skin sections were fixed in Formalin, embedded in paraffin, and cut into multiple sections of 5-µm thickness. Sections were mounted on slides, then the sections were dehydrated and defatted. The slides were then dip-coated with Kodak NTB3 photographic emulsion and allowed to expose for 20 days. After the exposure period, slides were developed and fixed in Kodak D-19 developer and Kodak Rapidfix, respectively. Sections were stained with Mayer's hemotoxylin and eosin, then examined and photographed using a Nikon Microfot-FX microscope. For all tracers, multiple sections from all three individuals treated were observed and photographed.

Redistribution of the radiolabels during the processing of the tissue samples may have affected the localization of the labeling in this study. This is, however, unlikely because a previous study that used fluorescent labels and the same methods of tissue processing showed no differences in the distribution of the fluorescent tracers when compared with samples prepared by cryomicrotomy (Stout and Ruth, 1998). However, it is possible that small amounts of the radiolabels were lost during tissue processing, which may have resulted in reduced sensitivity.

	Results

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Figures 1 and 2 present examples of the patterns seen for each of the tracers. All hair follicles pictured were in anagen or the growth phase of the hair development cycle. All images are of follicles and hair forming structures as indicated in figure legends. The anatomy of mouse hair follicles is discussed in detail in Stout and Ruth (1998) and Hojiro (1972). Good development of a latent image was obtained for all of the tracers except for ³⁶Cl. Previous results (Stout et al., 1998b) showed that ³⁶Cl could be easily removed from the mature hair by even mild aqueous conditions. Thus, it is likely that this tracer was lost from the sample sections during processing.

View larger version (122K): [in this window] [in a new window]   Fig. 1.   Microautoradiography of tracer deposition in C57 mice. The white arrow indicates the direction of hair growth. A, untreated C57 mouse showing background silver grain formation (bar is 25 µm). Black arrow indicates a silver grain. B, low magnification of silver deposition from a C57 mouse 6 h after a [35S]cysteine dosage. Arrows delineate the band of silver grain formation (bar is 25 µm). C, follicle from a C57 mouse 5 min after [35S]cysteine showing histologic detail, including hair papilla (Hp) and glassy membrane (Gm) (bar is 50 µm). D, incident lighting of the same frame showing profuse and diffuse silver grain formation over all cells in the papilla, indicating cysteine deposition in all cells. E, follicle from a C57 mouse 5 min after a 45Ca2+ dosage. Black arrow indicates melanosomes (bar is 25 µm). F, incident lighting of the same frame showing silver grain formation primarily over the melanosomes and melanocytes, indicating Ca2+ deposition primarily in the melanosomes. G, follicle from a C57 mouse 5 min after a [14C]urea dosage showing histologic detail (bar is 50 µm). H, incident lighting of the same frame showing diffuse formation of silver grains over all cells in the follicle, indicating urea deposition in all cells.

View larger version (111K): [in this window] [in a new window]   Fig. 2.   Microautoradiography of tracer deposition in BALB/c mice. A white arrow indicates the direction of hair growth. A, follicle from an untreated BALB/c mouse showing background formation of silver grains (bar is 25 µm). B, low magnification of silver deposition from a BALB/c mouse 6 h after a [35S]cysteine dosage. Arrows delineate the band of silver grain formation (bar is 25 µm). C, follicle from a BALB/c mouse 5 min after [35S]cysteine showing histologic detail, including hair papilla (Hp); the frame is of a follicle in cross-section (bar is 25 µm). D, incident lighting of the same frame showing profuse and diffuse silver grain formation over all cells in the papilla, indicating cysteine deposition in all cells. E, follicle from a BALB/c mouse 5 min after a 45Ca2+ dosage (bar is 25 µm). F, incident lighting of the same frame, showing sparse silver grain formation unlike that seen in C57 mice. G, follicle from a BALB/c mouse 5 min after a [14C]urea dosage, showing histologic detail (bar is 50 µm). H, incident lighting of the same frame showing diffuse formation of silver grains over all cells in the follicle, indicating urea deposition in all cells.

Figure 1 is composed of photomicrographs of skin sections from C57 mice. Panel A (original magnification, 600×) is of an untreated mouse with the hair papilla clearly visible (Hp). The black arrow indicates a silver grain and the white arrow indicates the direction of hair growth in all of the following figures. The background development of silver grains was very low. Panel B is a low magnification (original magnification, 40×) picture of several full-length longitudinal sections of hairs 6 h after a [³⁵S]cysteine dose. The black arrows delineate a band of silver grains deposited over maturing hair that contains [³⁵S]cysteine that has grown from the bottom of the hair bulb. This pattern is consistent with the growth of hair and was evident with all of the tracers. Panels C and D are of a C57 follicle at higher magnification (original magnification, 600×) 5 min after a dose of [³⁵S]cysteine. Panel C is a bright field picture showing histologic detail, and panel D is with incident lighting that shows the diffuse deposition of [³⁵S]cysteine throughout the follicle.

Panels E and F are of a C57 mouse 5 min after a dose of ⁴⁵Ca²⁺ (original magnification, 1200×). Panel E is a brightfield picture showing histologic detail. Panel F shows a patchy ⁴⁵Ca²⁺ labeling, which is located primarily over melanocytes/melanosomes. At 6 and 24 h postdose, ⁴⁵Ca²⁺ deposition was evident farther up the hair follicle, and was similar to that observed for cysteine.

Panels G and H are of a C57 mouse 5 min after a [¹⁴C]urea dose (original magnification, 600×). Panel G is a brightfield picture showing histologic detail. Panel H shows diffuse [¹⁴C]urea labeling over the entire follicle. At 6 and 24 h, [¹⁴C]urea deposition was evident farther up the hair follicle as was seen with cysteine.

Figure 2 is composed of micrographs of skin sections from BALB/c mice. Panel A (original magnification, 600×) is a follicle from an untreated animal showing minimal background silver grain development and was similar to that observed in control C57 mice. Panel B is a low magnification (original magnification, 40×) picture of a longitudinal section of a BALB/c hair follicle 6 h after a [³⁵S]cysteine dose. The black arrows delineate a band of [³⁵S]cysteine labeling over maturing hair that has grown from the bottom of the hair bulb. This pattern is the same as that seen in the C57 animals. Panels C and D are of a BALB/c mouse hair follicle at higher magnification (original magnification, 1200×) 5 min after a [³⁵S]cysteine dose. Panel C is a brightfield image showing histologic detail, and panel D is with incident lighting and shows diffuse [³⁵S]cysteine labeling throughout the follicle. A white arrow is not present, as this is a cross-section of the follicle; however, the hair papilla is visible (Hp). Again, the pattern of [³⁵S]cysteine localization is the same as that seen in the C57 animals.

Panels E and F are of a BALB/c mouse 5 min after a dose of ⁴⁵Ca²⁺ (original magnification, 1200×). Panel E is a brightfield picture showing histologic detail. Panel F shows sparse ⁴⁵Ca²⁺ labeling that is similar to background levels.

Panels G and H are of skin sections taken from a BALB/c mouse 5 min after a dose of [¹⁴C]urea (original magnification, 600×). Panel G is a brightfield image showing histology. Panel H shows [¹⁴C]urea labeling over the entire follicle. At 6 and 24 h, [¹⁴C]urea labeling was evident farther up the hair follicle and was similar to that seen with cysteine.

	Discussion

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The results of this study are consistent with results we reported previously using the same systemic tracer administration and measuring the concentrations in mature hair from C57 and BALB/c mice by liquid scintillation counting (Stout et al., 1998a). In that study, liquid scintillation counting (LSC) showed that the cation Ca²⁺ was found in greater concentrations in mature C57 hair than it was in BALB/C hair. The current histological results (Fig. 1, E and F and Fig. 2, E and F) indicate that ⁴⁵Ca²⁺ is associated with melanin in the forming hair. The incorporation of Ca²⁺ was resistant to removal during processing of the sample sections, which was also consistent with previous findings of Ca²⁺ resistance to removal by several aqueous extraction methods (Stout et al., 1998b). Enochs et al. (1997) reported that melanin has a high affinity for Ca²⁺, and Potts and Au (1976) also reported that higher atomic weight cations have a higher affinity for melanin. Consistent with this, we observed relative hair concentrations in C57 mice of Ca²⁺ > Mg²⁺ > Na⁺ (Stout et al., 1998b). These results suggest that Ca²⁺ is tightly bound by melanin and is not easily displaced.

The deposition pattern of [¹⁴C]urea was also consistent with previous findings that demonstrated similar concentrations in both C57 and BALB/c mature hair by LSC (Stout et al., 1998b). As can be seen in Fig. 1, G and H and Fig. 2, G and H, [¹⁴C]urea was deposited throughout the cells of the papilla in both C57 and BALB/c mice. The data suggest that urea is incorporated into the keratinocytes of the forming hair, but not into the melanosomes or melanocytes. The data also suggest that the protein component of hair is capable of neutral, lipophilic interactions and that neutral drugs may incorporate into hair in a more pigment-independent fashion.

The incorporation of cysteine into hair was also consistent with previous findings that demonstrated similar concentrations of labeled cysteine in mature hair from BALB/c and C57 mice (Stout et al., 1998b). Figure 1, C and D and Fig. 2, C and D demonstrate cysteine deposition throughout cells of the papilla in both C57 and BALB/c mice, and indicate that cysteine is incorporated into keratinocytes, consistent with cysteine being a structural component of hair proteins.

These results also demonstrated that single doses of tracer compounds could be resolved as distinct bands within the hair (Figs. 1B and 2B). This is consistent with other findings in our laboratory using systemically administered fluorescent tracers (Stout and Ruth, 1998).

These results support a multicompartmental model of drug incorporation into hair. Drugs and other compounds may partition between lipophilic compartments, primarily associated with the hair proteins and ion-exchangeable compartments primarily in the melanin. The capacity for melanin to participate in both lipophilic and hydrophilic interactions may exceed the nonpigment-dependent deposition in the protein compartment. Thus the possibility for pigment-dependent deposition and the confounding of results due to hair coloration may continue to present a problem for the utility of hair testing.