Hyperprolactinemia modifies extracellular matrix components associated with collagen fibrillogenesis in harderian glands of non- and pregnant female mice.

The harderian gland (HG) is a gland located at the base of the nictating membrane and fills the inferomedial aspect of the orbit in rodents. It is under the influence of the hypothalamic-pituitary-gonadal axis and, because of its hormone receptors, it is a target tissue for prolactin (PRL) and sex steroid hormones (estrogen and progesterone). In humans and murine, the anterior surface of the eyes is protected by a tear film synthesized by glands associated with the eye. In order to understand the endocrine changes caused by hyperprolactinemia in the glands responsible for the formation of the tear film, we used an animal model with metoclopramide-induced hyperprolactinemia (HPRL). Given the evidences that HPRL can lead to a process of cell death and tissue fibrosis, the protein expression of small leucine-rich proteoglycans (SLRPs) was analyzed through immunohistochemistry in the HG of the non- and the pregnant female mice with hyperprolactinemia. The SRLPs are related to collagen fibrillogenesis and they participate in pro-apoptotic signals. Our data revealed that high prolactin levels and changes in steroid hormones (estrogen and progesterone) can lead to an alteration in the amount of collagen, and in the structure of type I and III collagen fibers through changes in the amounts of lumican and decorin, which are responsible for collagen fibrillogenesis. This fact can lead to the impaired functioning of the HG by excessive apoptosis in the HG of the non- and the pregnant female mice with HPRL and especially in the HG of pregnancy-associated hyperprolactinemia.

Morphological and molecular changes in the Harderian gland of streptozotocin-induced diabetic rats.

Using a rat model of type 1 diabetes (T1D) obtained by treatment with streptozotocin, an antibiotic that destroys pancreatic ß-cells, we evaluated the influence of subsequent hyperglycemia on the morphology and physiology of the Harderian gland (HG). HG is located in the medial corner of the orbit of many terrestrial vertebrates and, in rodents, is characterized by the presence of porphyrins, which being involved in the phototransduction, through photo-oxidation, produce reactive oxygen species activating the autophagy pathway. The study focused on the expression of some morphological markers involved in cell junction formation (occludin, connexin-43, and α-tubulin) and mast cell number (MCN), as well as autophagic and apoptotic pathways. The expression of enzymes involved in steroidogenesis [steroidogenic acute regulatory protein (StAR), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD)] and the level of lipid peroxidation by thiobarbituric acid reactive species assay were also evaluated. The results strongly indicate, for the first time, that T1D has a negative impact on the pathophysiology of rat HG, as evidenced by increased oxidative stress, morphological and biochemical alterations, hyperproduction and secretion of porphyrins, increased MCN, reduced protein levels of StAR and 3ß-HSD, and, finally, induced autophagy and apoptosis. All the combined data support the use of the rat HG as a suitable experimental model to elucidate the molecular damage/survival pathways elicited by stress conditions.

High-fat diet affects autophagy and mitochondrial compartment in rat Harderian gland.

The Harderian gland (HG) of Rattus norvegicus is an orbital gland secreting lipids that accumulate in excess under condition of increased lipid metabolism. To study the response elicitated by lipid overload in rat HG, we housed the animals in thermoneutral conditions (28-30°C) in association to high fat diet (HFD). In HFD rats alterated blood lipid levels result in lipid accumulation in HG as demonstrated by the increased gland weight and histochemical/ultrastructural analyses. The HFD-caused oxidative stress forces the gland to trigger antioxidant defense mechanisms and autophagic process, such as lipophagy and mitophagy. Induction of mitochondrial DNA (mtDNA) damage and repair was stronger in HFD-rat HGs. An increase in marker expression levels of mitochondrial biogenesis, fission, and fusion occurred to counteract mtDNA copy number reduction and mitophagy. Therefore, the results demonstrate that rat HG activates autophagy as survival strategy under conditions of increased lipid metabolism and suggest a key role for mitophagy and membrane dynamics in the mitochondrial adaptive response to HFD.

Preliminary study of the ameliorative effects of melatonin on cadmium-induced morphological and biochemical alterations in the rat Harderian gland.

Herein is reported, for the first time in the rat Harderian gland (HG), the counteractive action of melatonin (Mlt), a well-known antioxidant radical scavenger, on the increased oxidative stress damages induced by a pro-oxidant substance, cadmium (Cd), an environmental pollutant also considered as endocrine disruptor. HG, an infraorbital gland present in almost all terrestrial vertebrates, produces a lipid secretion to lubricate the eyeball, as well as porphyrin/Mlt as light transducers. Moreover, HG is an extra-gonadal source of steroid sex hormones. Via ex vivo experiments lasting for 24 h, we verified the increased lipid peroxidation in Cd-treated glands, producing morphological alteration of the glandular epithelium, as well as an increased porphyrins accumulation. Moreover, Cd also induced a decreased protein level of the steroidogenic enzymes steroidogenic acute regulatory (StAR) and 3ßHSD, and an increased mast cell number. Results obtained with Mlt cotreatment demonstrated that it decreased the levels of Cd-induced oxidative damage, with reversal of all the observed modification. Furthermore, the TUNEL assay showed that the increased number of apoptotic cells in Cd-treated HG was counteracted by the contemporaneous Mlt administration. Results confirmed that Mlt treatment restored the levels of two autophagy markers, LC3 and p62, counteracting the autophagy Cd-induced. Interestingly, the positive effects of Mlt alone were highlighted by the decreased rate of lipid peroxidation as compared with the control, confirming its antioxidant action. Combined data further confirmed the antioxidant action of Mlt in counteracting the degeneration provoked by reactive oxygen species (ROS) in the rat HG, a tissue extremely susceptible to oxidative stress condition.

Dry eye symptoms in interferon regulatory factor 3-deficient mice due to herpes simplex virus infection in harderian gland and lacrimal gland.

PURPOSE: Dry eye syndrome (DES) is a multifactorial ocular disorder. The possible pathogens and pathogenic mechanisms for virus-related dry eye disease are largely unknown. The current study aimed to provide evidence for mechanisms contributing to DES induced by herpes simplex virus (HSV) infection in the harderian gland (HG) and lacrimal gland (LG). METHODS: We recorded the dry eye-like cornea pathology of irf3-/- mice infected with HSV-1 till 8 months of age. The slit-lamp and confocal microscopy was used to observe the corneal defects. TUNEL was used to detect the corneal apoptosis. Human corneas suffered from herpes stromal keratitis (HSK) were also analyzed as a comparison. Then, we measure the aqueous tear production with a phenol red thread test in irf3-/-mice, and recorded their tear film breakup time. HGs and LGs were sectioned and analyzed using HE and oil-red-O staining. For molecular signaling pathway analysis, we used mRNA sequencing to explore the related gene ontology. Western blotting (WB) and real-time reverse transcription-quantitative polymerase chain reaction were used to verify the level of the Akt signaling pathway and related inflammatory factors. RESULTS: Inoculated irf3-/- mice tended to develop dry eye-like symptoms, such as corneal keratinization, corneal cell apoptosis, and tear reduction. The HGs and LGs of irf3-/- mice showed increased level of HSV-1, and exhibited inflammatory pathological changes and impaired function, which explained the damaged tear film. WB and mRNA sequencing indicated that enhanced PI3K-Akt pathway in irf3-/- mice might account for the higher susceptibility to HSV infection. CONCLUSIONS: We observed evidence of DES in irf3-/- mice induced by HSV-1 infection in the HGs and LGs, which may introduce a potential novel target for DES treatment.

Hamster DAX1: Molecular insights, specific expression, and its role in the Harderian gland.

DAX1 plays an essential role in the differentiation and physiology of the Hypothalamic-Pituitary-Adrenal-Gonadal (HPAG) axis during embryogenesis. However, in adult tissues, in addition to the HPAG axis, evidence has not been found for its differential expression and function. We isolated the DAX1 cDNA to analyze its tissue localization and gene expression profiles in male and female hamsters' Harderian glands (HGs), Mesocricetus auratus. The isolated cDNA clone contains 1848 base pairs (bp), and a 1428-bp open reading frame (ORF) encodes a 476 amino acid protein. Sequence alignments and the phylogenetic tree display a relevant percentage of similarity with human (66%), rat (81%), and mouse (84%) sequences. In adult tissues, the mRNA distribution demonstrated that DAX1 is present in testis, ovaries, and male and female HGs. The highest expression profiles were identified in the adrenal glands, where females exhibit higher mRNA levels than males. The sexually dimorphic expression of DAX1 in adrenals suggests that its presence could be associated with regulating, functioning, and maintaining this endocrine tissue. These findings indicate that the DAX1 gene is limitedly expressed in adult tissues. In the HGs, we demonstrate the absence of sexually dimorphic gene expression. Our results suggest that DAX1 might have an additional physiological function outside of the HPAG axis, specifically in the HG, which may be required for the regulation of intracrine steroidogenesis, secretion, and maintenance of exocrine tissue.

Novel Lipids of the Rabbit Harderian Gland Improve Tear Stability in an Animal Model of Dry Eye Disease.

Purpose: Instability of the tear film leads to evaporative dry eye disease (EDED), but the Harderian gland in some terrestrial vertebrates may produce novel lipids that stabilize the tear film and protect against dry eye. Here, the nonpolar lipids in the Harderian gland and tears of the rabbit but absent in human tears were identified and tested in preclinical studies to determine whether they could treat severe EDED. Methods: Lipids were identified primarily by atmospheric pressure chemical ionization mass spectrometry (MS) and fragmentation MS/MS. An identified lipid was synthesized and formulated as an emulsion and as a cyclodextrin (CD) clathrate. Following doses with test agents and controls, tear film breakup time (TBUT), tear production, corneal fluorescein staining, macrophage infiltration, and goblet cell survival were measured using standard tests at 0, 2 and 4 weeks in an animal model of EDED. Results: The lipid emulsion increased TBUT (P < 0.01) and tear production (P < 0.05), while it decreased corneal staining (P < 0.01) compared to controls. The lipid CD formulation increased TBUT (P < 0.05) and tear production (P < 0.05) but had no significant effect on the remaining test parameters. There were no differences in macrophage infiltration and conjunctival impression cytology scores between the formulations and their vehicle controls. Conclusions: Lipids in the rabbit Harderian gland and tears differ from those identified in human meibum and tears. These unique rabbit lipids may confer a protective effect against EDED and, as supplements to human tears, fulfill a similar role.

Selective autophagy, lipophagy and mitophagy, in the Harderian gland along the oestrous cycle: a potential retrieval effect of melatonin.

Sexual dimorphism has been reported in many processes. However, sexual bias in favour of the use of males is very present in science. One of the main reasons is that the impact of hormones in diverse pathways and processes such as autophagy have not been properly addressed in vivo. The Harderian gland is a perfect model to study autophagic modulation as it exhibits important changes during the oestrous cycle. The aim of this study is to identify the main processes behind Harderian gland differences under oestrous cycle and their modulator. In the present study we show that redox-sensitive transcription factors have an essential role: NF-κB may activate SQSTM1/p62 in oestrus, promoting selective types of autophagy: mitophagy and lipophagy. Nrf2 activation in dioestrus, leads the retrieval phase and restoration of mitochondrial homeostasis. Melatonin's receptors show higher expression in dioestrus, leading to decreases in pro-inflammatory mediators and enhanced Nrf2 expression. Consequently, autophagy is blocked, and porphyrin release is reduced. All these results point to melatonin as one of the main modulators of the changes in autophagy during the oestrous cycle.

Expression and regulation of ABCG2/BCRP1 by sex steroids in the Harderian gland of the Syrian hamster (Mesocricetus auratus).

The ATP-Binding Cassette, subfamily G, member 2 (ABCG2) transporter is associated with the regulation of protoporphyrin IX transport and of other intermediates in heme biosynthesis. Because the hamster Harderian gland (HG) exhibits high concentrations of porphyrins and sexual dimorphism, we analyzed the hamster ABCG2. Cloned cDNA [2098-base pairs (bp)] contains an open-reading frame (ORF) of 1971-bp that encodes a 656 amino-acid protein with a molecular weight of 72844.56Da. The hamster ABCG2 sequence is conserved phylogenetically and shares a high percentage of identity with mouse (89%), rat (88%), and human (84%) transporters. Within its structure, a Walker A (G-X-X-G-X-G-K-S), a C signature motif characteristic of ABC transporters, and six putative transmembrane domains (TMDs) were identified. ABCG2 mRNA was detected in all hamster tissues, with higher amounts found in HG, brain, cerebellum, kidney, gut, ovary, and testis. Harderian ABCG2 expression exhibits a sexually dimorphic pattern where females display higher mRNA levels than males. Different patterns of transcriptional profiles of ABCG2 during the estrous cycle and after gonadectomy in both sexes were also observed. The differential expression between male and female HGs suggests that ABCG2 is under the regulation of gonadal steroids. The ABCG2 transporter is likely involved in the endogenous regulation of porphyrins in hamster HGs.

Isolation and characterization of abundantly-expressed cDNAs from the Harderian gland of the garter snake (Thamnophis sirtalis: Colubridae).

The Harderian gland (HG) is an orbital structure whose proteinaceous secretions pass through the nasolacrimal duct to the vomeronasal organ (VNO). Though these three structures occur in many tetrapod vertebrates, the garter snake (Thamnophis sirtalis) is one of the few vertebrates in which the passage of the proteinaceous secretions have been experimentally shown. Secreted proteins from the HG may function as transporters for chemical signals to the VNO epithelium. To investigate the proteins being produced by the HG of the garter snake, cDNA libraries were constructed from HG mRNA, and several individual cDNAs were analyzed by sequencing, RT-qPCR, and PCR on genomic DNA. Two of the three cDNAs that were characterized are abundantly expressed only in the Harderian gland and contain putative signal sequences for secretion, which makes them candidates for transporter proteins secreted from the HG. One is a member of the large lipocalin family of proteins, based on its similarity to other members of that protein family. Many lipocalins are binding/carrier proteins for a variety of ligands. The other is a family of proteins, with five members identified so far, all of unknown structure and function and present in the garter snake genome but not in other squamate genomes.

Isolation and sex steroid effects on the expression of the ATP-binding cassette transporter ABCB6 in Harderian glands of hamster (Mesocricetus auratus).

ATP-Binding Cassette, subfamily B, member 6 (ABCB6) is a transporter that is upregulated by elevated intracellular porphyrin concentrations. In the Harderian gland (HG), the synthesis of porphyrins appears to be under the influence of gonadal steroids and to exhibit a dimorphic pattern. To explore whether ABCB6 is also influenced by sex steroids, we isolated its specific cDNA sequence and investigated its mRNA levels in the HGs of hamsters. ABCB6's cDNA sequence presents an open reading frame (ORF) of 2529 bp that encodes a predicted 842-amino acid (aa) protein with a molecular weight of 93 kDa. Multiple sequence alignments showed that ABCB6's aa sequence is highly conserved and shares the highest homology (93%) with mouse ABCB6. RT-qPCR analysis indicated that ABCB6 is expressed in all the tissues examined, exhibiting high expression levels in the liver, adrenal glands, and testis. The mRNA concentrations of ABCB6 in HGs were very similar between males and in females; similarly, gonadectomy and treatment with sex steroids appear to scarcely affect ABCB6 mRNA levels. The intraglandular content of ABCB6 mRNA showed discrete, though non-significant, variations through the estrous cycle. The results provide evidence that gonadal steroids have a minimal physiological role on the regulation of ABCB6 expression and might indicate that this transporter has a small effect on porphyrin trafficking in the HGs of hamsters.

Structural study and expression of the androgen receptors during the reproductive cycle in the Harderian gland of the male Meriones libycus.

The goal of this study was to evaluate for the first time the expression of the androgen receptors (AR) in Harderian glands (HG) of the male Meriones lybicus in relation to the reproductive cycle. Six male Harderian glands of the resting period and 6 of the breeding period were collected. The animals were trapped in the desert of Béni Abbès (Algeria). The morphology of the Harderian glands was studied by light microscopy and morphometry, whereas the expression of the androgen receptors was assessed and quantified based on immunohistochemistry techniques. We have shown that the Harderian glands of Meriones libycus are tubuloalveolar glands with wide lumen. The glandular epithelium is composed of two types of cells (types I and II) in the resting season and three types of cells (types I, II and III) in the breeding season. These three types of cells differ in size and shape. Type-I cells have a prismatic shape, an acidophilic cytoplasm, and small lipidic vacuoles, whereas type-II ones are pyramidal in shape, with basophilic cytoplasm. Type-III cells resemble those of type I, and so they are prismatic in shape and have an acidophilic cytoplasm with larger lipidic vacuoles. The immunoreactivity of type-I and type-III cells was mainly cytoplasmic and the intensity of the immunostaining was significantly higher during the breeding season. Among other functions, the Harderian gland seems to be involved in the production of pheromones under the effect of androgens.

The Harderian gland transcriptomes of Caraiba andreae, Cubophis cantherigerus and Tretanorhinus variabilis, three colubroid snakes from Cuba.

The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100 bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ.

Molecular regulation of ocular gland development.

The tear film is produced by two ocular glands, the lacrimal glands, which produce the aqueous component of this film, and the meibomian glands, which secrete the lipidic component that is key to reduce evaporation of the watery film at the surface of the eye. Embryonic development of these exocrine glands has been mostly studied in mice, which also develop Harderian glands, a third type of ocular gland whose role is still not well understood. This review provides an update on the signalling pathways, transcription factors andextracellular matrix components that have been shown to play a role in ocular gland development.

Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain.

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1ß, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-ß gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Novel analysis of the Harderian gland transcriptome response to Newcastle disease virus in two inbred chicken lines.

Behind each eye of the chicken resides a unique lymph tissue, the Harderian gland, for which RNA sequencing (RNA-seq) analysis is novel. We characterized the response of this tissue to Newcastle disease virus (NDV) in two inbred lines with different susceptibility to NDV across three time points. Three-week-old relatively resistant (Fayoumi) and relatively susceptible (Leghorn) birds were inoculated with a high-titered (107EID50) La Sota strain of NDV via an oculonasal route. At 2, 6, and 10 days post infection (dpi) Harderian glands were collected and analyzed via RNA-seq. The Fayoumi had significantly more detectable viral transcripts in the Harderian gland at 2 dpi than the Leghorn, but cleared the virus by 6 dpi. At all three time points, few genes were declared differentially expressed (DE) between the challenged and nonchallenged birds, except for the Leghorns at 6 dpi, and these DE genes were predicted to activate an adaptive immune response. Relative to the Leghorn, the Fayoumi was predicted to activate more immune pathways in both challenged and nonchallenged birds suggesting a more elevated immune system in the Fayoumis under homeostatic conditions. Overall, this study helped characterize the function of this important tissue and its response to NDV.

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland.

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology.

Comparative study of phototoxicity of protoporphyrin IX synthetic and extracted from ssp Rattus novergicus albinus rats toward murine melanoma cells.

Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.

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.

Synergy Theory in Radiobiology.

Customized open-source software is used to characterize, exemplify, compare and critically evaluate mathematical/computational synergy analysis methods currently used in biology, and used or potentially applicable in radiobiology. As examples, we reanalyze some published results on murine Harderian gland tumors and on in vitro chromosome aberrations induced by exposure to single-ion radiations that simulate components of the galactic cosmic ray field. Baseline no-synergy/no-antagonism-mixture dose-effect relationships are calculated for corresponding mixed fields. No new experimental results are presented. Synergy analysis of effects due to a mixed radiation field whose components' individual dose-effect relationships are highly curvilinear should not consist of simply comparing to the sum of the components' effects. Such curvilinearity must often be allowed for in current radiobiology, especially when studying possible non-targeted ("bystander") effects. Consequently, many different synergy analysis theories are currently used in biology to replace simple effect additivity. We give evidence that for most synergy experiments and observations, incremental effect additivity is the most appropriate replacement. It has a large domain of applicability, being useful even when pronounced individual dose-effect relationship curvilinearity is a confounding factor. It allows calculation of 95% confidence intervals for baseline mixture dose-effect relationships taking into account parameter correlations; if non-targeted effects are important this gives much tighter intervals than neglecting the correlations. It always obeys two consistency conditions that simple effect additivity usually fails to obey: a "mixture of mixtures principle" and the standard "sham mixture principle". The mixture of mixtures principle is important in radiobiology because even nominally single-ion radiations are usually mixtures when they strike the biological target, due to intervening material. It is not yet clear whether mixing galactic cosmic ray components sometimes leads to statistically significant synergy for animal tumorigenesis. The substantial limitations of synergy theories are sometimes overlooked, and they warrant further study.