The in vivo effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on N-methyl-N-nitrosourea-induced apoptosis in mouse hair follicles.

This study assessed the in vivo relationship between apoptosis induced by the tumor initiator N-methyl-N-nitrosourea (MNU) and action of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse hair follicle matrix cells. Mouse hair follicles were stimulated to grow hair synchronously by plucking resting hairs and MNU was applied to the plucked skin as the apoptosis inducer. The effects of TPA on MNU-induced apoptosis, when given at different intervals before or after MNU treatment, were examined. Changes in the percentage of apoptotic cells among total hair matrix cells after TPA treatment were measured. A significant suppression in levels of MNU-induced apoptosis was observed in the animals receiving TPA 1 to 6 hr following the induction. Administration of TPA before MNU caused a reduction in numbers of apoptotic cells over the control groups, but the differences were not significant. Determination of the diurnal variation in apoptotic levels in vehicle-treated mouse hair follicles revealed a relatively constant baseline pattern, suggesting that the above apoptotic responses to MNU and TPA were not affected by the background levels of apoptosis. The findings provided in vivo evidence which would support the hypothesis that TPA promotes tumorigenesis by preventing carcinogen-initiated cells from undergoing apoptotic death.

Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 1. Follicle morphogenesis in wild-type mice.

The morphogenesis of hairs is initiated and maintained by reciprocal interactions between groups of epithelial and mesenchymal cells. To examine whether cell adhesion molecules play a role in this process, prenatal distribution patterns of various cell adhesion molecules were studied during hair follicle morphogenesis in the dorsal skin of C57BL mouse embryos, using monoclonal antibodies. E-cadherin was present on all epithelial cells of the skin when the ectoderm gave rise to periderm and epidermis. E-cadherin was reduced in the follicle placodes and hair plugs, then disappeared from the presumptive hair matrix of elongating follicles. P-cadherin was initially present on all cells of periderm and epidermis and was later retained at a reduced level in the basal epidermal layer. P-cadherin was prominent in all follicle placodes and hair plugs and in the presumptive hair matrix of elongating follicles. N-CAM was present on all mesenchymal cells of the presumptive dermis at the prefollicle stage, then temporarily restricted to a few cells just below the dermal-epithelial junction. Later, N-CAM reappeared in the interfollicular mesenchyme and was prominent in the mesenchymal sheath and dermal papilla of elongating follicles. In addition, N-CAM was expressed in the hair plugs, then became progressively restricted to the upper caudal part of the elongating follicles. The results suggest that the main role of cell adhesion molecules is to mould the follicle by relaxing or reinforcing cell contacts in areas of increased morphogenetic activity.

Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 2. Follicle morphogenesis in the hair mutants, Tabby and downy.

Wild-type mice have three main types of hair in their pelage: tylotrichs, awls and zigzags. Tabby mice have a yellowish coat consisting of awls only, whereas downy mice have a sparse grayish coat consisting of unusually fine hairs. The spatial and temporal distribution of cell adhesion molecules (CAMs) during hair follicle morphogenesis was investigated in the mutants and compared with that in nonmutant mice. In Tabby embryos, awl follicles developed normally and showed normal immunostaining patterns for E-cadherin, P-cadherin and N-CAM. Prior to follicle initiation, however, some deviations from normal skin morphology and staining patterns indicated a delay in the development of the basal epidermal layer. On the other hand, the stratum corneum was formed prematurely. Therefore, the lack of tylotrich and zigzag follicles in Tabby mice might be explained by a general defect in epidermal development rather than by abnormal CAM expression. In downy embryos, tylotrich and awl follicles were initiated within the normal time periods, but elongation and differentiation of most follicles were abnormal. At birth, most follicles were small and/or severely deformed but showed normal CAM expression patterns. Extreme distortion and disorientation of follicles seemed to be associated with disintegration of the dermal papilla and abnormal mesenchymal cell condensations between the follicles. This suggests that abnormal hair development in downy mice might result from a defect in dermal rather than epidermal components of the skin.

Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8.

In order to explore the origin and significance of Merkel cells in the hairy skin of mammals, the development of Merkel cells and nerve endings in the dorsolateral skin of C57BL mouse embryos was studied in serial cryostat sections. At 13 and 14 days of gestation, application of a monoclonal antibody to mouse keratin 8 (mK8) resulted in specific immunofluorescence of all cells in the epidermis and periderm. The periderm retained specific staining until it was shed, around 18 days. At 15 days, mK8-specific staining elsewhere was restricted to scattered immature Merkel cells in the developing tylotrich follicles and the adjacent epidermis. Between 16 and 17 days, these cells assembled within the basal epidermal layer, caudal to each tylotrich follicle, to form a disc-shaped rudiment of a 'haarscheibe' or touch dome. No Merkel cells were found in association with the later developing awl and zigzag follicles. In mice homozygous or hemizygous for the Tabby mutation, in which tylotrich follicles never form, no Merkel cells were found in any part of the dorsolateral skin. In mice homozygous for the recessive downy mutation, in which all three types of hair are present but reduced in size, Merkel cell development was the same as in wild-type mice. Nerve endings were located in the upper dermal mesenchyme by a monoclonal antibody to neural cell adhesion molecule. This antibody also stained plasma membranes in specific parts of the hair follicles during their development. From 14 to 19 days, none of the nerve endings were seen in contact with the epidermis or the follicle epithelium, even in areas where Merkel cells were located. These findings support the view that both location and early differentiation of Merkel cells in the dorsolateral epidermis are independent of neural influences but linked to the development of tylotrich follicles.

Mechanism of recovery in esophageal epithelia of rats with severe zinc deficiency.

Forty-six rats fed a Zn-deficient diet for 18 days, were divided into three groups and treated with Zn-deficient diet (GI), a normal (Zn-adequate) diet (GII) or a pharmacological Zn therapy diet (GIII) for 3 days. For light and electron microscopy, samples were taken at times 0, 8, 12, 24 and 72 h after treatment. In treatment GI, at all times, all rats had esophageal parakeratosis. With treatment GII, there was a variable progression toward normalization of the epithelium at 12 and 24 h. At 72 h there was almost complete recovery of normal epithelium. In treatment GIII at 8 h, large, light cells in the basal layer were shown to be present between dark cylindrical cells, a finding which was transitionary and disappeared at 12 h. Also, a thin keratinized layer was observed above the granular layer at 12 h. Recovery had progressed at 24 h and was complete after 72 h. The results are discussed in terms of a potential role of Zn in the sequence of cytochemical events in epithelial differentiation.

The secret life of the hair follicle.

The mammalian hair follicle is a treasure waiting to be discovered by more molecular geneticists. How can a tiny cluster of apparently uniform epithelial cells, adjacent to a tiny cluster of uniform mesenchymal cells, give rise to five or six concentric cylinders, each of which is composed of cells of a distinctive type that synthesize their own distinctive set of proteins? There is now evidence that several growth factors, cell adhesion molecules and other molecules play important roles in the regulation of this minute organ.

A new type of lesion associated with severe fur damage in Canadian ranch foxes and an investigation of possible causes.

In the silver fox, as in its wild ancestor, the red fox (Vulpes vulpes L.), the annual growing phase (anagen) of guard hair follicles occupies at least four months. Severe damage to the hair coat near the end of this growing period was reported in 1985 on many ranches in New Brunswick and Nova Scotia. A histological analysis of serial sections of skin biopsies showed a marked increase in nuclear aberrations in the hair matrix of anagen guard hair follicles. These nuclear aberrations indicated that cells were undergoing apoptosis, a controlled form of cell death. Tissues from affected and unaffected foxes for histological and toxicological analysis, as well as other data, were obtained during visits to 26 ranches in 1986 and 34 ranches in 1987. Histological sections of the 1987 skin samples showed the mean percentage of nuclear aberrations in 43 unaffected foxes to be 0.08 +/- 0.01 (SEM), while that for 49 affected foxes was 0.51 +/- 0.23. The four foxes with the most severe coat damage also had the highest incidences of guard hair matrix cells with nuclear aberrations, ranging from 20 to 100 times greater than the mean for unaffected foxes. The mitotic index of the hair matrix, which normally remains fairly constant during the hair growth phase, was similar for unaffected and affected foxes (1.83 +/- 0.06 and 1.97 +/- 0.07 respectively). Although our analyses of field data have not established a specific environmental factor associated with increased nuclear aberrations, the possible involvement of toxic agents in follicle damage may warrant further investigation.

Either chick embryo dermis or retinoid-treated mouse dermis can initiate glandular morphogenesis from mammalian epidermal tissue.

Excess retinoids can cause developing mouse vibrissa follicles to be transformed into mucous glands in organ culture. The objective was to test the hypothesis that retinoids act in this system by altering morphogenetic properties of the dermis. After inititation by retinoic acid (RA) in organ culture, glands were shown to develop further in embryonic skin grafted to the chick chorioallantoic membrane (CAM). Recombinants of 12.5 day mouse epidermis with untreated or RA-treated mouse or chick dermis were then grafted to CAM for 7 days. For homospecific recombinants, 13.5 day mouse dermis originated from 11.5 day skin cultured for 2 days, with or without 5.2 microgram/ml RA. For heterospecific recombinants, 12 day dermis came from chick embryos, previously injected with 250 microgram RA. Glands were absent from the homospecific recombinants including untreated mouse dermis, but appeared in 26% of those with RA-treated dermis. Among heterospecific recombinants, 75% of those with RA-treated chick dermis and 29% of those with untreated dermis had glands. Untreated 10-12 day chick skin contained two forms of endogenous vitamin A, retinol (4.5 microgram/g protein) and dehydroretinol (3.7 microgram/g protein), while 13-14 day mouse skin contained only retinol (1.8 microgram/g protein), as shown by high performance liquid chromatography. RA injection increased retinol and dehydroretinol in chick skin, while RA was undetectable. Thus RA can act through mouse dermis to form epithelial glands and through chick dermis to increase the incidence of glands. The glands in recombinants with untreated chick dermis may result from the higher levels of endogenous retinoids in chick skin, compared with mouse skin.

Comparison of the activity of topically applied pesticides and the herbicide 2,4-D in two short-term in vivo assays of genotoxicity in the mouse.

Genotoxicity of eight topically applied compounds was determined using the bone marrow micronucleus (MN) test and hair follicle nuclear aberration (NA) assay in CD1 mice. Twenty-four hours after a single treatment, cyclophosphamide (CY), applied at doses corresponding to 1/4, 1/8, 1/16, and 1/32 of the published dermal LD50, and N-methyl-N-nitrosourea (MNU), applied at 1/4, 1/8, and 1/16 of the published dermal LD50, were found to increase the incidence of NA in a dose-dependent manner. The frequency of MN was significantly increased only at the highest dose of CY. Using the same protocol, six pesticides applied in dimethyl sulfoxide (DMSO) at doses of 1/8, 1/16, and 1/32 of the dermal LD50 were investigated. Aminocarb and chlordane induced a dose-dependent increase in the frequency of NA, while there was an observed increase in NA incidence at only the highest doses of dichlorvos (DDVP), 4,4'-DDT (DDT), and 2,4-dichlorophenoxyacetic acid (2,4-D). No effect was observed with fenitrothion on nuclear aberrations in hair follicles. Except for the highest dose of chlordane, none of the pesticides tested positive in the bone marrow micronucleus test. Serum cholinesterase levels were reduced to 70 +/- 4.7% of the DMSO control level with DDVP, 57 +/- 8.2% with aminocarb, and 60.3 +/- 4.8% with fenitrothion, indicating some systemic activity with these topically applied agents. The data suggest that aminocarb, chlordane, DDVP, DDT, and 2,4-D are genotoxic as determined by the NA assay and that this assay may be more useful in detecting topically applied genotoxic agents than the more often used bone marrow micronucleus test.

Persistence of added retinoids in organ culture media during induction of mucous metaplasia and glandular morphogenesis in hamster cheek pouches.

The retinoid concentration (determined colorimetrically) did not change significantly in retinyl acetate-supplemented (6 micrograms/ml) Eagle's Minimal Essential Medium containing 10% fetal calf serum when stored at -20 or 4 degrees C over 7 days. After the medium was incubated at 37 degrees C for 48 h, 37-49% of the retinoid remained, whether or not tissue (neonatal Syrian hamster cheek pouch) was present, and irrespective of explant age. The normal retinoid level in the tissue was approximately 0.25 micrograms per gram. Therefore, neonatal hamster cheek pouches, incubated in medium with the addition of 6 micrograms of retinyl acetate per ml of medium and undergoing mucous metaplasia and some mucous gland morphogenesis, were continually being exposed to retinoid levels which, though gradually decreasing, remained well above their normal physiological level.

Excess retinoid acts through the stroma to produce mucous glands from newborn hamster cheek pouch in vitro.

Many morphogenetic processes are modified or initiated by retinoids. The cheek pouch of the newborn hamster can be induced to develop mucous glands in vitro by adding excess retinoid. The objective of this study was to determine whether the retinoid acted through the epithelium or the mesenchymal stroma. Explants of cheek pouch were grown for 7 days in either standard medium, or medium supplemented with 6 micrograms/ml retinyl acetate (RAc; 1.8 x 10(-5) M). After separation of most explants into epithelium and mesenchyme by trypsinization, the separated tissues were recombined in all possible ways and cultured for a further 1-2 weeks in standard medium. All explants were analysed histologically and/or histochemically from complete serial paraffin sections. No glands were formed in 30 recombinants containing stroma that had not been exposed to RAc, but four of 25 recombinants containing previously exposed stroma had glands, as well as four of 18 unseparated explants exposed to RAc. Exposure of epithelium to RAc did not result in the incidence of glands. It was concluded that RAc acting through the stroma was responsible for the instructive interaction with the epithelium for gland formation. A molecular mechanism is suggested.

Nuclear aberrations in hair follicle cells of patients receiving cyclophosphamide. A possible in vivo assay for human exposure to genotoxic agents.

The toxic effect of cyclophosphamide on the proliferative cell population of hair follicles plucked from the human scalp was examined by the in vivo nuclear aberration assay. Patients participating in an independent clinical trial received oral low dose cyclophosphamide, intravenous high dose cyclophosphamide or oral placebo treatment. The percent of cells with nuclear aberrations (indicating apoptosis, a special form of cell death) and the percent of mitotic cells, in the hair matrix, were calculated for each patient before treatment and at several time points following cyclophosphamide or placebo treatment. The mean percentages of nuclear aberrations in both the treated Low dose and High dose cyclophosphamide patients were significantly higher than those for the pre-treatment and Placebo patients. The nuclear aberrations in hair follicle cells increased from pre-treatment (and Placebo) to treated Low dose and finally to treated High dose patients. The average percentage for pre-treatment samples from all patients was 0.06 +/- 0.03 SE. For 1 week and 1 month samples from Low dose patients it was 0.35 +/- 0.08 SE, and for combined 2,3 and 4 day samples from High dose patients it was 1.08 +/- 0.12 SE. Cyclophosphamide also had a significant effect on mitosis. A decrease in mitotic activity was observed at 1 month following the initial low dose cyclophosphamide treatment and at 24 +/- 2 h following each of the first two high dose cyclophosphamide treatments. The observed increase in nuclear aberrations following low dose as well as high dose cyclophosphamide suggests that it is feasible to use the nuclear aberration assay for in vivo human genotoxicity testing, using proliferating hair follicle cells.

The use of retinoids as probes for analyzing morphogenesis of glands from epithelial tissues.

Thirty-five years ago Honor Fell and Edward Mellanby were studying effects of high doses of vitamin A on skeletal development in chick embryos when they noticed that a piece of epidermis, accidentally included in an organ culture, had undergone mucous metaplasia. Further studies by Fell and others eventually led to an understanding of the important role of vitamin A in modulating epithelia in vivo. Fifteen years later another organ culture experiment showed me that excess vitamin A could also initiate the morphogenesis of branching and mucus-secreting glands from developing vibrissa follicles in upper lip skin of embryonic mice. Since then our group has shown that induction of this novel structure by naturally occurring retinoids resembles a normal embryonic induction in that it is stage-dependent, time-dependent, and irreversible. Tissue separation and recombination studies showed that isolated upper lip epidermis can form these glands when combined with retinoid-treated upper lip dermis. Untreated mouse epidermis can form similar glands after combination with chick dermis containing higher retinoid levels. The hamster cheek pouch, normally devoid of glandular structures, can also form mucous glands when treated with a retinoid, either in vivo or in vitro. Recombination studies in organ culture have now shown that mesenchyme exposed to retinoid is essential for gland morphogenesis from pouch epithelium. Evidence is accumulating that retinoic acid may even be the active morphogen in some normally developing systems.

An inherited connective tissue disease in the horse.

The hyperextensible, fragile skin of two related horses was compared with the skin of eight normal horses. Skin sections were examined by light microscopy and transmission electron microscopy. The deep dermal layer of the dorsal abdomen was much thinner in the affected horses, and contained bundles of collagen fibers which were more loosely packed. Within individual fibers, the fibrils were frequently curved and nonparallel rather than straight and parallel. Both of the affected animals had a greater range of fibril diameters than a normal horse. They had some unusually thick fibrils with very irregular outlines in cross-sections, not observed in the normal animal. Other skin samples were subjected to acetic acid extraction, pepsin digestion, amino acid analysis and polyacrylamide gel electrophoresis. In the skin of the two affected horses, the proportion of total extracted collagen which was acid-soluble was twice as high as in two normal horses. Collagen types I and III were present in similar proportions in normal and affected horses, and the collagen chains were of normal molecular weights. The disorder resembles the group described by Minor (Minor RR: Am J Pathol 98: 226, 1980) as 'dominant collagen packing defect I' which has been reported in dogs, mink, and cats, and which shares features with Ehlers Danlos Syndrome I, II, and III in man. The pedigree data available for these horses suggest an autosomal recessive mutation, but are also consistent with autosomal dominant inheritance.

Stability of the glandular morphogenesis produced by retinoids in the newborn hamster cheek pouch in vitro.

Retinoids can induce alterations in differentiation and morphogenesis in the hamster cheek pouch. In order to determine the stability of these changes, explants of neonatal pouch were exposed to 6 micrograms/ml of either retinyl acetate (RAc: 1.8 x 10(-5) M) or all-trans retinoic acid (RA: 2.0 x 10(-5) M) for an initial 3 of 7 days, out of a total of 21 days in organ culture. Three days of RAc or RA caused a delay in the differentiation and keratinization of the epithelium at least up to day 7 of culture. Additionally, two out of ten explants exposed to RA showed small downgrowths of epithelium into the stroma at 7 or 14 days. Seven days of exposure to either retinoid led to inhibition of epithelial keratinization, and produced a mucous metaplasia which was still seen at the end of the 21-day culture period. Periodic acid-Schiff (PAS)-positive, diastase-resistant material was present in the metaplastic epithelium, in intercellular, and in some instances, intracellular locations. An excess of either RAc or RA, for 7 days, induced persistent glandlike downgrowths of epithelium, suggesting that a stable alteration in the developmental program of the epithelium may have occurred. Many of these downgrowths possessed a lumen which was lined by cuboidal epithelium and contained PAS-positive, diastase-resistant secretory material. RA appeared more potent than RAc in inhibiting keratinization, in producing a mucous metaplasia, and in initiating glandlike downgrowths. The persistence of glandular downgrowths suggests that retinoids, either directly or indirectly, act in a manner similar to that of an embryonic inductor.

Normal levels of vitamin A in tissues of the Syrian hamster (Mesocricetus auratus) determined colorimetrically.

Vitamin A levels in tissues of 20 normal adult hamsters on a standard diet were measured colorimetrically. No significant difference between male and female animals was found for any of the tissues sampled. The mean vitamin A value for blood plasma in 20 animals was 53.4 micrograms/dl. Mean values for liver, kidneys, flank skin and cheek pouch were 813, 1.29, 1.84 and 1.31 mg/g wet weight, respectively. The vitamin assay was less suitable for small organs such as trachea.

Effects of intradermally injected and topically applied mouse epidermal growth factor on wool growth, skin and wool follicles of merino sheep.

Twice daily intradermal (ID) injections of mouse epidermal growth factor (mEGF) in sterile saline for 1-4 days into delineated areas of skin of Merino sheep produced dose-dependent changes in wool follicles and fibres, ranging from slight reduction in follicle bulb size and transient disturbance of cuticle formation on some fibres to the induction of catagen of follicles and shedding of fibres with distorted, tapered ends. Regeneration of follicles commenced by day 7. By contrast, ID injections of saline did not affect follicle activity. The epidermis became thicker and more parakeratotic after multiple injections of mEGF than after injection of saline, but was almost normal again by day 14. Persistent small increases in sebaceous gland size, additional to those induced by ID injections of saline, and delayed small increases in sweat gland size also occurred after multiple injections of mEGF. Daily topical applications of mEGF in 50% (v/v) aqueous propylene glycol 5 days each week for 4 weeks did not affect wool growth or the follicles and other skin components. The only effect observed, due to application of the aqueous propylene glycol, was an increase in the number of layers of cornified cells in the stratum corneum of the epidermis, with the cells arranged in clearly discernible stacks. The effects produced by ID injections of mEGF indicate that mEGF acts directly on the pilosebaceous and epidermal components of skin.