Induction and phosphorylation of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in the rat retina upon optic nerve injury

Several eye diseases are associated with axonal injury in the optic nerve, which normally leads to degeneration of retinal ganglion cells (RGCs) and subsequently to loss of vision. There is experimental evidence that some members of the small heat shock protein family (HspBs) are upregulated upon optic nerve injury (ONI) in the retina and sufficient to promote RGC survival. These data raise the question as to whether other family members may play a similar role in this context. Here, we performed a comprehensive comparative study comprising all HspBs in an experimental model of ONI. We found that five HspBs were expressed in the adult rat retina at control conditions but only HspB1 and HspB5 were upregulated in response to ONI. Furthermore, HspB1 and HspB5 were constitutively phosphorylated in Müller cells at serine 15 and serine 59, respectively. In RGCs, phosphorylation was stimulated by ONI and occurred at serine 86 of HspB1 and at serine 19 and 45 of HspB5. These data suggest that of all small heat shock proteins, only HspB1 and HspB5 might be of protective value for RGCs after ONI and that this process might be regulated by phosphorylation at serine 86 of HspB1 and serine 19 and serine 45 of HspB5. The molecular targets of phosphoHspB1 and phosphoHspB5 remain to be identified.

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The online version of this article (doi:10.1007/s12192-015-0650-8) contains supplementary material, which is available to authorized users.     

Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in cultured hippocampal neurons

The so-called stress response involving upregulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia or oxidative stress. To gain more information about the molecular targets by which HspB1 (Hsp25) and HspB5 (αB-crystallin) might exert their neuroprotective effect we investigated the subcellular localization of unphosphorylated and phosphorylated HspB1 and B5 in neurons by immunocytochemistry and subcellular fractionation. In cultured hippocampal neurons, the unphosphorylated forms of both Hsps were localized in the perikaryon and nucleus, whereas the phosphorylated forms were recruited into neuronal processes. pHspB1-Ser15 and -Ser 86 were found within dendrites with a punctate distribution pattern partially colocalizing with the synaptic marker vGlut-1. pHspB5-Ser19 and -Ser45 localized to axons and dendrites with a filamentous-like staining pattern, whereas pHspB5-Ser59 was found in dendrites, especially along the plasma membrane and in spines. Biochemical analysis, i.e. subcellular fractionation of rat brain with subsequent Western blotting supported these localizations. These data show that in neurons HspB1 and B5 may have various molecular interaction partners at synapses, within dendrites and axons and that this interaction is likely to be regulated by phosphorylation. Stress-induced phosphorylation of HspB1 and B5 may lead to binding of these Hsps to their targets at synapses and neuronal processes which might provide one important mechanism of how they exert their neuroprotective effect.     

Disruption of NMDAR–CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca²⁺ channels. Building on our discovery of the interaction and regulation of Ca²⁺ channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca²⁺ channel. Remarkably, we also found that this region attenuated Ca²⁺ influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2''s role in excitotoxicity and neuroprotection.     

Disruption of NMDAR–CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca²⁺ channels. Building on our discovery of the interaction and regulation of Ca²⁺ channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca²⁺ channel. Remarkably, we also found that this region attenuated Ca²⁺ influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.     

Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.     

Piperine suppresses cerebral ischemia–reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-κB in middle cerebral artery occlusion rat model

The pathophysiological mechanisms leading to neuronal injury in middle cerebral artery occlusion (MCAO) model of cerebral stroke are complex and multifactorial that form the bases of behavioral deficits and inflammation mediated damage. The present study demonstrates the effect of piperine pretreatment (10 mg/kg b wt, once daily p.o. for 15 days) on cerebral ischemia-induced inflammation in male Wistar rats. The right middle cerebral artery was occluded for 2 h followed by reperfusion for 22 h. A maximum infarct volume (57.80 %) was observed in ischemic MCAO group. However, piperine administration prior to ischemia showed a significant reduction in infarct volume (28.29 %; p < 0.05) and neuronal loss (12.72 %; p < 0.01). As a result of piperine pretreatment, a significant improvement in behavioral outputs of MCAO rats (p < 0.05-0.01) was observed. Piperine successfully reduced the level of proinflammatory cytokines IL-1β, IL-6 and TNF-α, in ischemic group (p < 0.01). Ischemic group brain has shown edematous morphology with vacuolated architecture and pyknotic nuclei in H & E staining which was successfully ameliorated by piperine administration. Moreover, piperine also succeeded in lowering the expression of COX-2, NOS-2, and NF-κB (p < 0.01). Both cytosolic and nuclear NF-κB were down-regulated in ischemic group pre-administered with piperine (p < 0.01). The present study suggests that piperine is able to salvage the ischemic penumbral zone neurons by virtue of its anti-inflammatory property, thereby limiting ischemic cell death.     

1α,25-dihydroxyvitamin D(3) mechanism of action: modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats

The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D(3) (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10 day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca(2+) uptake and potassium and chloride channels. Also, the Na(+)/K(+)-ATPase inactivation by ouabain mimicked the 1,25D action on (45)Ca(2+) uptake. The Na(+)/K(+)-ATPase inhibition observed herein might lead to intracellular Na(+) accumulation with subsequent L-VDCC opening and consequently increased (45)Ca(2+) (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP-dependent protein kinase (PKA), Ca(2+)/calmodulin-dependent protein kinase (PKCaMII), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase p38 (p38(MAPK)). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.     

Small heat shock proteins HSP27 (HspB1), αB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death

Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Dexmedetomidine alleviates cerebral ischemia-reperfusion injury in rats via inhibition of hypoxia-inducible factor-1α

Dexmedetomidine (Dex) was reported to reduce ischemia-reperfusion (I/R) injury in kidney and brain tissues. Thus, we aimed to study the role and mechanism of Dex in cerebral I/R injury by inhibiting hypoxia-inducible factor-1α (HIF-1α) and apoptosis. First, I/R injury models were established. Six groups were assigned after different treatments: sham, I/R, I/R+Dex, I/R+2-methoxyestradiol (2ME2) (HIF-1α inhibitor), I/R+CoCl ₂ (HIF-1α activator), and I/R+Dex+CoCl ₂ groups. Neurological function, cerebral infarction volume, survival, and apoptosis of brain cells were then analyzed. Besides, immunohistochemistry and Western blot analysis were used to detect the expression of HIF-1α, BCL-2[B-cell leukemia/lymphoma 2] adenovirus E1B interacting protein 3 (BNIP3), B-cell leukemia/lymphoma 2 (BCL2), BCL2[B-cell leukemia/lymphoma 2] associated X (Bax), and cleaved-caspase3 proteins in brain tissues. I/R rats showed cerebral infarction, increased neurological function score, number of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)-positive cells and HIF-1α–positive cells as well as decreased neurons. Inhibition of HIF-1α can reduce the apoptosis induced by I/R, and overexpression of HIF-1α can aggravate apoptosis in brain tissue of I/R rats. Furthermore, activation of HIF-1α expression blocks the inhibitory effect of Dex on neuronal apoptosis in I/R rats. Dex may inhibit the neuronal apoptosis of I/R rats by inhibiting the HIF-1α pathway and then improve the cerebral I/R injury in rats.     

Mutants of human αB-crystallin cause enhanced protein aggregation and apoptosis in mammalian cells: Influence of co-expression of HspB1

Mutations of human αB-crystallin cause congenital cataract and cardio-myopathy by protein aggregation and cell death. How mutations of αB-crystallin become pathogenic is poorly understood. To better understand the cellular events related to protein aggregation and cell death, we transfected cataract and cardio-myopathy causing mutants, R11H, P20S, R56W, D109H, R120G, D140N, G154S, R157H and A171T in HeLa cells and assessed protein aggregation and apoptosis by laser scanning confocal microspy (LSCM) and flow cytometry. Cells individually transfected with the mutants, D109H, R120G, D140N and R157H significantly showed more aggregates. Cells overexpressed with HspB1 (Hsp27) significantly sequestered aggregates in all mutants and suppressed apoptosis in mutants, P20S, D109H and A171T. Significant increases of apoptotic cells as stained with Annexin V were observed in mutants, D109H and A171T transfected cells. Cells positive for active caspase-3 was increased in the mutant, D109H. Thus the previously recognized anti-apoptotic functions of αB-crystallin were compromised in these mutants.     

Immunocytochemical localization of 5α-reductase type 1 in the prostate of normal and castrated rats

We immunohistochemically studied the localization of 5-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.     

Catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats by targeting HIF-1α/VEGF

BackgroundThe initial factor in the occurrence, development, and prognosis of cerebral ischemia is vascular dysfunction in the brain, and vascular remodeling of the brain is the key therapeutic target and strategy for ischemic tissue repair. Catalpol is the main active component of the radix of Rehmannia glutinosa Libosch, and it exhibits potential pleiotropic protective effects in many brain-related diseases, including stroke.PurposeThe present study was designed to investigate whether catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats and to identify its possible mechanisms in vivo and in vitro.Study designCerebral ischemic rats and oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to study the therapeutic potential of catalpol in vivo and in vitro.MethodsFirst, neurological deficits, histopathological morphology, infarct volume, vascular morphology, vessel density, and angiogenesis in focal cerebral ischemic rats were observed to test the potential treatment effects of catalpol. Then, oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to mimic the pathological changes in vessels during ischemia to study the effects and possible mechanisms of catalpol in protecting vascular structure and promoting angiogenesis.ResultsThe in vivo results showed that catalpol reduced neurological deficit scores and infarct volume, protected vascular structure, and promoted angiogenesis in cerebral ischemic rats. The in vitro results showed that catalpol improved oxygen-glucose deprivation-induced damage and promoted proliferation, migration, and in vitro tube formation of brain microvascular endothelial cells. The HIF-1α (hypoxia-inducible factor 1α)/VEGF (vascular endothelial growth factor) pathway was activated by catalpol both in the brains of cerebral ischemic rats and in primary brain microvascular endothelial cells, and the activating effects of catalpol were inhibited by SU1498.ConclusionThe results of both the in vivo and in vitro studies proved that catalpol protects vascular structure and promotes angiogenesis in focal cerebral ischemic rats and that the mechanism is dependent on HIF-1α/VEGF.     

Steroid 5α-reductase type 1 immunolocalized in the anterior pituitary of intact and castrated male rats

The localization of 5α-reductase was immunohistochemically studied in the anterior pituitary of male rats, using a polyclonal antibody against 5α-reductase rat type 1. The immunoreactive cells were concentrated in the central region and on the border of the intermediate lobe in the anterior pituitary, but not in the intermediate or posterior lobe. The immunoreaction was located mostly in the cytoplasm and occasionally in the cell nuclei. The immunoreactive cells showed alterations in size and number and in the intensity of the immunoreaction after gonadectomy. One week after castration, the cells became larger and the immunoreactivity increased. Two weeks after castration, the number of immunoreactive cells increased. Double immunostaining using antiluteinizing hormone β-subunit or anti-follicle stimulating hormone β-subunit antibody revealed that most of the cells containing 5α-reductase were gonadotrophs. Electron microscopically, the immunoreactive cells showed lamelliform rough endoplasmic reticulum and a depletion of secretory granules 1 week after castration. One week later, the rough endoplasmic reticulum was developed and dilated and the number of secretory granules increased. These results suggest that 5α-reductase is located in the gonadotrophs of rat anterior pituitary and that it is involved in the feedback regulation of gonadotropin secretion by androgens.     

Time-course alterations of Toll-like receptor 4 and NF-κB p65, and their co-expression in the gerbil hippocampal CA1 region after transient cerebral ischemia

Innate immune system is very important to modulate the host defense against a large variety of pathogens. Toll-like receptors (TLRs) play a key role in controlling innate immune response. Among TLRs, TLR4 is a specific receptor for lipopolysaccharide and associated with the release of pro-inflammatory cytokines. In the present study, we investigated ischemia-related changes of TLR4 immunoreactivity and its protein level, and nuclear factor κB (NF-κB) p65 immunoreactivity regarding inflammatory responses in the hippocampal CA1 region after 5 min of transient cerebral ischemia to identify the correlation between transient ischemia and inflammation. In the sham-operated group, TLR4 immunoreactivity was easily detected in pyramidal neurons of the hippocampal CA1 region (CA1). TLR4 immunoreactivity in pyramidal neurons was distinctively decreased after ischemia/reperfusion (I/R); instead, based on double immunofluorescence study, TLR4 immunoreactivity was expressed in non-pyramidal neurons and astrocytes from 2 days postischemia. In addition, TLR4 protein level was lowest at 1 day postischemia and highest 4 days after I/R. On the other hand, NF-κB p65 immunoreactivity was not detected in the CA1 of the sham-operated group, and NF-κB p65 immunoreactivity was not observed until 1 day after I/R. However, NF-κB p65 immunoreactivity began to be expressed in astrocytes at 2 days postischemia, and the immunoreactivity was strong 4 days postischemia. Our results indicate that TLR4 and NF-κB p65 immunoreactivity are changed in CA1 pyramidal neurons and newly expressed in astrocytes, not in microglia, in the CA1 region after transient cerebral ischemia.     

HDAC1/2-mediated regulation of JNK and ERK phosphorylation in bovine mammary epithelial cells in response to TNF-α

Bovine mammary epithelial cells (MAC-Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC-T cells stimulated with tumor necrosis factor α (TNF-α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF-α-mediated inflammatory gene expression. Of note, we report that HDAC1/2-mediated inflammatory gene expression was partly regulated by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2-specific inhibitors may prove efficacious for the treatment of bovine mastitis.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.