Immunomodulatory effect of ginseng stem-leaf saponins and selenium on Harderian gland in immunization of chickens to Newcastle disease vaccine.

Our previous study demonstrated that ginseng stem-leaf saponins (GSLS) in combination with selenium (GSLS-Se) have adjuvant effect on the live vaccine of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) in intraocular-and-intranasal immunization in chickens. The present study was to investigate the potential molecular mechanisms involved in the immunomodulation of GSLS-Se on the Harderian gland (HG). It was found that the window allowing animals susceptible to infections due to low antibody titers became smaller or even completely closed because of increased NDV-specific HI titers when NDV vaccine and GSLS-Se were coadministered for immunization at early life in chickens. In addition, NDV-specific sIgA and the numbers of IgG+, IgA+, IgM+ plasma cells were significantly more in GSLS-Se group than the control in the HGs. Transcriptome analysis of HGs identified 1184 differentially expressed genes (DEGs) between GSLS-Se treated and non-treated groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified 42 significantly enriched GO terms and 13 canonical immune pathways. These findings indicated that GSLS-Se might exert immunomodulatory effects through influencing the antioxidant regulation and modulating the activity of immune related enzymes. Besides, Toll-like receptor (TLR) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway might be involved primarily in the immunomodulation. Therefore, enhanced antibody responses in GSLS-Se group may be attributed to the immunomodulatory effects of GSLS-Se on the immune-related gene profile expressed in the immunocompetent cells of the HGs.

Transcriptional profiling of male CD-1 mouse lungs and Harderian glands supports the involvement of calcium signaling in acrylamide-induced tumors.

Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA-carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA-sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2-year cancer bioassay) and lungs of AA-exposed male CD-1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw-day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw-day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA-induced mouse carcinogenicity, which is consistent with a recently proposed CS-based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans.

Pharmacokinetics, Distribution, Metabolism, and Excretion of Omadacycline following a Single Intravenous or Oral Dose of 14C-Omadacycline in Rats.

The absorption, distribution, metabolism, and excretion (ADME) of omadacycline, a first-in-class aminomethylcycline antibiotic with a broad spectrum of activity against Gram-positive, Gram-negative, anaerobic, and atypical bacteria, were evaluated in rats. Tissue distribution was investigated by quantitative whole-body autoradiography in male Long-Evans Hooded (LEH) rats. Following an intravenous (i.v.) dose of 5 mg/kg of body weight, radioactivity widely and rapidly distributed into most tissues. The highest tissue-to-blood concentration ratios (t/b) were observed in bone mineral, thyroid gland, and Harderian gland at 24 h post-i.v. dose. There was no evidence of stable accumulation in uveal tract tissue, suggesting the absence of a stable binding interaction with melanin. Following a 90 mg/kg oral dose in LEH rats, the highest t/b were observed in bone mineral, Harderian gland, liver, spleen, and salivary gland. The plasma protein binding levels were 26% in the rat and 15% to 21% in other species. Omadacycline plasma clearance was 1.2 liters/h/kg, and its half-life was 4.6 h; the steady-state volume of distribution (Vss) was 6.89 liters/kg. Major circulating components in plasma were intact omadacycline and its epimer. Consistent with observations in human, approximately 80% of the dose was excreted into the feces as unchanged omadacycline after i.v. administration. Fecal excretion was primarily the result of biliary excretion (∼40%) and direct gastrointestinal secretion (∼30%). However, urinary excretion (∼30%) was equally prominent after i.v. dosing.

Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

Class B CpG ODN stimulation upregulates expression of TLR21 and IFN-γ in chicken Harderian gland cells.

This study aimed to evaluate the response of Harderian gland (HG) cells after in vitro stimulation with class B synthetic oligodeoxyribonucleotides (ODN) containing CpG motifs. This knowledge is of importance for the development of mucosal vaccines for poultry, such as eye-drop or spray vaccines, to determine if class B CpG ODN can act as an vaccine adjuvant or as a prophylactic treatment mainly against respiratory disease viruses. The relative expression of Toll-like receptor 21 (TLR21), interferon (IFN)-γ, interleukin (IL)-1ß and IL-10 genes were quantified at 1, 3, 6 and 18 h post-stimulation of HG cells from 5-week-old birds. In addition, it was also investigated if expression of these genes was affected by the age of the birds (differences between 5- and 12-week-old birds), concentrations of ODN or cell preparation method used. Class B CpG ODN induced upregulation of TLR21 and IFN-γ mRNA expression levels at 1h post-stimulation depending on concentration of ODN used but only in HG cells isolated from young birds.

Dorzolamide cyclodextrin nanoparticle suspension eye drops and Trusopt in rabbit.

PURPOSE: Dorzolamide nanoparticle γ-cyclodextrin eye drops may prolong the effect of dorzolamide on intraocular pressure. We test whether the nanoparticle drops have an irritating or toxic effect on the eye in an in vivo rabbit model. METHODS: Eighteen pigmented rabbits were divided into 4 groups receiving dorzolamide nanoparticle γ-cyclodextrin eye drops×1/day or×2/day, Trusopt® (dorzolamide HCl)×3/day, and untreated controls that received no drops. The rabbits received treatment for 1 month. After sacrifice, 33 eyes and 25 Harderian glands were evaluated for histopathology in a masked way. RESULTS: Mild inflammation was seen in 19/31 eyes and 13/23 Harderian glands. The difference in inflammation (n=eyes/n=glands)between the γ-cyclodextrin nanoparticle eye drops×1/day (n=5/5),×2/day (n=5/3), Trusopt (n=7/4), or untreated control (n=2/0) groups was nonsignificant in both eyes and glands (P=0.87 and P=0.92) Acute inflammation was seen in 1 Harderian gland that received γ-cyclodextrin nanoparticle eye drops×2/day. The difference in conjunctival injection between the groups was nonsignificant (P=0.30). CONCLUSIONS: Dorzolamide γ-cyclodextrin nanoparticle eye drops are no more locally toxic or irritating to the eye than Trusopt.

Triiodothyronine induces lipid oxidation and mitochondrial biogenesis in rat Harderian gland.

The rat Harderian gland (HG) is an orbital gland producing a copious lipid secretion. Recent studies indicate that its secretory activity is regulated by thyroid hormones. In this study, we found that both isoforms of the thyroid hormone receptor (Trα (Thra) and Trß (Thrb)) are expressed in rat HGs. Although Thra is expressed at a higher level, only Thrb is regulated by triiodothyronine (T3). Because T3 induces an increase in lipid metabolism in rat HGs, we investigated the effects of an animal's thyroid state on the expression levels of carnitine palmitoyltransferase-1A (Cpt1a) and carnitine palmitoyltransferase-1B (Cpt1b) and acyl-CoA oxidase (Acox1) (rate-limiting enzymes in mitochondrial and peroxisomal fatty acid oxidation respectively), as well as on the mitochondrial compartment, thereby correlating mitochondrial activity and biogenesis with morphological analysis. We found that hypothyroidism decreased the expression of Cpt1b and Acox1 mRNA, whereas the administration of T3 to hypothyroid rats increased transcript levels. Respiratory parameters and catalase protein levels provided further evidence that T3 modulates mitochondrial and peroxisomal activities. Furthermore, in hypothyroid rat HGs, the mitochondrial number and their total area decreased with respect to the controls, whereas the average area of the individual mitochondrion did not change. However, the average area of the individual mitochondrion was reduced by ∼50% in hypothyroid T3-treated HGs, and the mitochondrial number and the total area of the mitochondrial compartment increased. The mitochondrial morphometric data correlated well with the molecular results. Indeed, hypothyroid status did not modify the expression of mitochondrial biogenesis genes such as Ppargc1a, Nrf1 and Tfam, whereas T3 treatment increased the expression level of these genes.

Inhalation carcinogenicity of 1,1,1-trichloroethane in rats and mice.

Carcinogenicity of 1,1,1-trichloroethane (TCE) was examined by an inhalation exposure of F344 rats and BDF1 mice of both sexes to TCE at 0, 200, 800 or 3200 ppm for 6 h/d, 5 d/week for 104 weeks. In male rats, the incidences of bronchiolo-alveolar adenomas and peritoneal mesotheliomas were significantly increased in the 800 and 3200 ppm-exposed groups, respectively. The incidence of bronchiolo-alveolar adenomas in the 3200 ppm-exposed groups exceeded the range of historical control data in the Japan Bioassay Research Center. In female rats, the tumor incidences were not increased in any organs of the TCE-exposed groups. In male mice, a significant positive trend with dose was shown for incidences of bronchiolo-alveolar carcinomas, combined incidences of bronchiolo-alveolar adenomas/carcinomas and hepatocellular adenomas. The incidence of Harderian gland adenomas was significantly increased in the 3200 ppm-exposed group, and malignant lymphomas of spleen at this highest dose exceeded the range of historical control data. In female mice, the combined incidence of bronchiolo-alveolar adenomas/carcinomas was significantly increased in the 3200 ppm-exposed group, and the incidences of hepatocellular adenomas and combined incidences of hepatocellular adenomas/carcinomas were significantly increased in the 200, 800 and 3200 ppm-exposed groups with dose dependence except the combined incidence of hepatocellular adenomas/carcinomas in the 200 ppm-exposed group. The incidences of bronchiolo-alveolar adenomas in the 3200 ppm-exposed group and combined incidences of hepatocellular adenomas/carcinomas in the 200 ppm-exposed groups exceeded the ranges of historical control data. Thus, this study provided clear evidence of inhalation carcinogenicity for TCE in both rats and mice.

Effects on high cholesterol-fed to liver, retina, hippocampus, and Harderian gland in Goto-Kakizaki rat.

To understand the relationship among cholesterolemia, hyperglycemic stage in non obese type 2 diabetes mellitus, and histological perturbations on liver, retina, hippocampus, and Harderian gland, we maintained rat on a diet high in cholesterol for fourteen weeks, then analyzed blood lipid profiles, blood glucose, hepatic enzymes, and microscopic lesion of those tissues. We observed that high cholesterol-treated rat elevated in cholesterol and low density lipoprotein with not correlated to hyperglycemia. Histopathological changing in Goto-Kakizaki rat on liver (microvesicular steatosis) and Harderain gland (tubular lesions) were related to hyperglycemic effect rather than cholesterolemic effect. These may be related to hypoinsulinemic characteristic of this diabetic model. However increasing pyknotic nuclei on hippocampus and reducing of retinal ganglionic cell were related to the high level of cholesterol loaded with synergized effect due to diabetic stage.

Carcinogenicity of acrylamide in B6C3F(1) mice and F344/N rats from a 2-year drinking water exposure.

Acrylamide is a component of roasted coffee and certain baked and fried carbohydrate-rich foods prepared at high temperatures. We have assessed the carcinogenicity of acrylamide in male and female B6C3F(1) mice and F344/N rats administered 0, 0.0875, 0.175, 0.35, or 0.70mM acrylamide in the drinking water ad libitum for 2 years. Acrylamide caused significant dose-related decreasing trends in the body weights of F344/N rats. Acrylamide administration resulted in significant dose-related decreasing trends in survival in both sexes of B6C3F(1) mice and in female F344/N rats. Histopathological analyses indicated significant dose-related increases in Harderian gland and lung tumors in male and female B6C3F(1) mice. Male B6C3F(1) mice also had a significantly increased incidence of forestomach tumors, while female B6C3F(1) mice had significant dose-related increases in mammary gland, ovary, and skin tumors. In male and female F344/N rats, there were significant increases in thyroid tumors. Male F344/N rats also had significant dose-related increases in testes, heart, and pancreas tumors, while female F344 rats demonstrated significant increases in clitoral gland, mammary gland, oral cavity, and skin tumors. These results, combined with previous mechanistic studies, provide strong support for the concept that acrylamide is activated to a carcinogen through metabolism to glycidamide.

Melatonin modulates autophagy through a redox-mediated action in female Syrian hamster Harderian gland controlling cell types and gland activity.

The Syrian hamster Harderian gland exhibits sexually dimorphic porphyrin biosynthesis, wherein the female glands display an extraordinarily high concentration of porphyrins. Damage derived from this production of porphyrins, mediated by reactive oxygen species, causes the glands to develop autophagic processes, which culminate in detachment-derived cell death; these cells normally play a central role in the secretory activity of the gland. The main aim of this study was to analyze how a change in the redox state impacts autophagy. Female Syrian hamsters were treated daily with melatonin (25 µg, subcutaneously) at ZT 10 for 1-2 months (N-acetyl-5-methoxytryptamine), an endogenous antioxidant that ameliorates the deleterious effects of free radicals via a variety of mechanisms. The length of treatment affected the redox balance, the autophagy machinery, and the activation of p53 and NF-κB. One-month treatment displaces redox balance to the antioxidant side, promotes autophagy through a p53-mediated mechanism, and increases cell detachment. Meanwhile, 2-month treatment restores redox balance to the oxidant side, activates NF-κB reducing autophagy to basal levels, increases number of type II cells, and reduces number of detached cells. Our results conclude that the redox state can modulate autophagy through redox-sensitive transcriptions factors. Additionally, these findings support a hypothesis that ascribes differences in the autophagic-lysosomal pathway to epithelial cell types, thereby restricting detachment-induced autophagic cell death to epithelial cell type I.

Effect of D-aspartate uptake on uncoupling protein-3 and α-tubulin expressions in rat Harderian gland.

Although D-aspartate (D-Asp) has been recognized as having an important physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we evaluated the oxidative stress response to D-Asp treatment in rat Harderian gland (HG) by measuring total cellular hydroperoxide levels. Further, we examined the effect of D-Asp uptake on the expression of the mitochondrial uncoupling protein-3 (UCP3), ß-actin, and α-tubulin. In rat HG, elevated levels of D-Asp significantly increased hydroperoxide production. This phenomenon was probably due to D-Asp uptake as well as lipid and porphyrin increased levels. Higher UCP3 levels and lower α-tubulin expression were also observed after D-Asp treatment. On the contrary, ß-actin expression was unchanged. Given the possible role of UCP3 in lipid handling, the higher expression of mitochondria UCP3 protein in D-Asp-treated HG may reflect a major need to export excessive amounts of hydroperoxides deriving from a greater fatty acid flux across these organelles and higher mitochondrial porphyrin levels. Moreover, abundance of hydroperoxides in D-Asp treated rat HG could determine the decrease of α-tubulin expression. Thus, our findings indicate that a high concentration of D-Asp is critical in initiating a cascade of events determined by oxidative stress.

Suppression of the later stages of radiation-induced carcinogenesis by antioxidant dietary formulations.

We have previously reported data from a long-term carcinogenesis study indicating that dietary antioxidant supplements can suppress radiation-induced malignant lymphoma and harderian gland tumors induced by space radiations (specifically, 1 GeV/n iron ions or protons) in CBA/J mice. Two different antioxidant dietary supplements were used in these studies: a supplement containing a mixture of antioxidant agents [l-selenomethionine (SeM), N-acetyl cysteine (NAC), ascorbic acid, co-enzyme Q10, α-lipoic acid and vitamin E succinate], termed the AOX supplement, and another supplement known as Bowman-Birk Inhibitor Concentrate (BBIC). In the present report, the results from the earlier analysis of the harderian gland data from the published long-term animal study have been combined with new data derived from the same long-term animal study. In the earlier analysis, harderian glands were removed from animals exhibiting abnormalities (e.g. visibly swollen areas) around the eyes at the time of euthanasia or death in the long-term animal study. Abnormalities around the eyes were usually due to the development of tumors in the harderian glands of these mice. The new data presented here focused on the histopathological results obtained from analyses of the harderian glands of mice that did not have visible abnormalities around the eyes at the time of necropsy in the long-term animal study. In this paper, the original published data and the new data have been combined to provide a more complete evaluation of the harderian glands from animals in the long-term carcinogenesis study, with all available harderian glands from the animals processed and prepared for histopathological evaluation. The results indicate that, although dietary antioxidant supplements suppressed harderian gland tumors in a statistically significant fashion when all glands were analyzed, the antioxidant diets were less effective at suppressing the incidence of all harderian gland tumors than they were at suppressing the incidence of large harderian gland tumors (>2 mm) observed at animal necropsy. These results suggest that the dietary antioxidant formulations had major suppressive effects in the later stages of radiation-induced carcinogenesis in vivo. It is hypothesized that the dietary antioxidant formulations prevented the early-stage neoplastic growths from progressing to fully developed, malignant tumors. In addition, the antioxidant dietary formulations were very effective at preventing the development of proton- or iron-ion-induced malignant tumors, because, in contrast to irradiated controls, no malignant tumors were observed in the irradiated animals maintained on either of the dietary antioxidant diets.

Molecular pathways involved in the cyclic activity of frog (Pelophylax esculentus) Harderian gland: Influence of temperature and testosterone.

The Harderian gland of Pelophylax esculentus (previously: Rana esculenta) shows seasonal secretory activity changes. Specifically, the secretory activity reaches a maximum during the hottest months, i.e., July and August, drops in September and slowly increases from October onwards. Expressions of P-CaMKII, P-ERK1 and P-Akt1 correlate well with gland secretory activity; i.e., they peak immediately before the hottest part of the year (maximum secretory activity). When the gland activity declines, kinase expressions drop and remain low until February. Experiments of thermal manipulation indicate that high temperature induces the activation of CaMKII, ERK1 and Akt1, and at low temperatures, Akt1 expression decreases. Experiments of chemical castration indicate that the Harderian gland of Cyproterone acetate-treated frogs shows lower Akt1 activity as compared to controls, but the CaMKII and ERK1 activities remain unchanged. Furthermore, in a period of resumed gland activity (October-December) we observed the highest expression of PCNA, a mitotic marker. Immediately after the proliferative phase, we found the highest expression of caspase 3, an enzyme that plays a key role in apoptosis. In combination, the results suggest the following: 1) CaMKII, ERK1, and Akt1 modulate the annual secretory activity of the frog Harderian gland; 2) CaMKII and ERK1 activities are regulated by temperature, whereas both temperature and testosterone likely play a central role in Akt1 regulation; and 3) proliferation and apoptosis occur to restore and balance, respectively, an adequate cell number, which is essential to gland function.

Two distinct types of mouse melanocyte: differential signaling requirement for the maintenance of non-cutaneous and dermal versus epidermal melanocytes.

Unlike the thoroughly investigated melanocyte population in the hair follicle of the epidermis, the growth and differentiation requirements of the melanocytes in the eye, harderian gland and inner ear - the so-called non-cutaneous melanocytes - remain unclear. In this study, we investigated the in vitro and in vivo effects of the factors that regulate melanocyte development on the stem cells or the precursors of these non-cutaneous melanocytes. In general, a reduction in KIT receptor tyrosine kinase signaling leads to disordered melanocyte development. However, melanocytes in the eye, ear and harderian gland were revealed to be less sensitive to KIT signaling than cutaneous melanocytes. Instead, melanocytes in the eye and harderian gland were stimulated more effectively by endothelin 3 (ET3) or hepatocyte growth factor (HGF) signals than by KIT signaling, and the precursors of these melanocytes expressed the lowest amount of KIT. The growth and differentiation of these non-cutaneous melanocytes were specifically inhibited by antagonists for ET3 and HGF. In transgenic mice induced to express ET3 or HGF in their skin and epithelial tissues from human cytokeratin 14 promoters, the survival and differentiation of non-cutaneous and dermal melanocytes, but not epidermal melanocytes, were enhanced, apparently irrespective of KIT signaling. These results provide a molecular basis for the clear discrimination between non-cutaneous or dermal melanocytes and epidermal melanocytes, a difference that might be important in the pathogenesis of melanocyte-related diseases and melanomas.

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance.

In this paper, the role of D-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous D-Asp were detected, along with aspartate racemases that convert D-Asp to L-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered D-Asp. D-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since D-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that D-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Melatonin and environmental lighting regulate ALA-S gene expression and So porphyrin biosynthesis in the rat harderian gland.

The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day-night PPIX levels as well as day-night activity and mode of expression of the porphyrinogenic enzymes delta-aminolevulinate synthase (ALA-S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630- to 670-fold higher than in the liver, without a day-night difference, which is the consequence of both low Fech gene expression (5- to 7-fold lower than in the liver) and ALA-S over-expression (4- to 7-fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day-night variation, whereas HG ALA-S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA-S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day-night ALA-S variation in the HG that is observed under standard 12 L:12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA-S gene expression in the rat HG.

Hormonal regulation and characterisation of the aldehyde oxidase-like gene of hamster Harderian gland.

The HG is a compound tubulo-alveolar gland located in the orbital cavity of the majority of vertebrates. In the golden hamster it shows a clear cut sexual dimorphism in both morphological and biochemical parameters such as cell types, protein pattern, lipid metabolism, porphyrin content, steroid hormone receptor expression. In a previous study we found that in primary culture of male hamster Harderian gland (HG), androgens (A) increase the MHG07 (male Harderian gland) expression and this effect is abrogated by both flutamide and cycloheximide. The present study represents a deeper analysis on MHG07 regulation by other members of steroid/thyroid hormone superfamily. Estrogens (E) impair the stimulatory effect of A and after the addition of a pure anti-estrogen, ICI 164,384, the negative effect of E is abrogated. Dexamethasone (Dex), used alone or in combination with A negatively affect the MHG07 expression. Also T(3) increases the expression of MHG07 mRNA. Progesterone (P) does not affect the expression of MHG07 mRNA. The use of cycloheximide abrogates the effect of steroids, suggesting that the latter act through their own receptors. Dose-response experiments show that low steroid concentrations (10(-12)M) are sufficient to affect the MHG07 expression. It is argued that the expression of MHG07 is under a highly coordinate relationship between androgen, estrogen, glucocorticoid, retinoic acid and thyroid hormones.

Dimorphic expression of uncoupling protein-3 in golden hamster harderian gland: effects of castration and testosterone administration.

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50 to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained alpha-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids.

K-ras mutations in lung tumors and tumors from other organs are consistent with a common mechanism of ethylene oxide tumorigenesis in the B6C3F1 mouse.

Ethylene oxide is a multisite carcinogen in rodents and classified as a human carcinogen by the National Toxicology Program. In 2-year mouse studies, ethylene oxide (EO) induced lung, Harderian gland (HG), and uterine neoplasms. We evaluated representative EO-induced and equivalent spontaneous neoplasms for K-ras mutations in codons 12, 13, and 61. K-ras mutations were identified in 100% (23/23) of the EO-induced lung neoplasms and 25% (27/108) of the spontaneous lung neoplasms. Codon 12 G to T transversions were common in EO-induced lung neoplasms (21/23) but infrequent in spontaneous lung neoplasms (1/108). K-ras mutations were found in 86% (18/21) of the EO-induced HG neoplasms and 7% (2/27) of the spontaneous HG neoplasms. Codon 13 G to C and codon 12 G to T transversions were predominant in the EO-induced HG neoplasms but absent in spontaneous HG neoplasms (0/27). K-ras mutations occurred in 83% (5/6) of the EO-induced uterine carcinomas and all were codon 13 C to T transitions. These data show a strong predilection for development of K-ras mutations in EO-induced lung, Harderian gland, and uterine neoplasms. This suggests that EO specifically targets the K-ras gene in multiple tissue types and that this event is a critical component of EO-induced tumorigenesis.