Persistence of the circadian rhythm of dehydroepiandrosterone in the brain, but not in the plasma, of castrated and adrenalectomized rats

In adrenalectomized and orchidectomized rats, the removal of the steroidogenic endocrine glands is associated with a near-disappearance of corticosterone (B) from plasma and brain; circadian variations of B and of plasma dehydroepiandrosterone (D), characteristic of the intact rat, are no more detected. By contrast, analyses of brain D measurements by the cosinor method demonstrate a persisting circadian rhythm of large amplitude.     

Neuro-steroids: 3 beta-hydroxy-delta 5-derivatives in rat and monkey brain

The rat brain accumulates pregnenolone (P) as the unconjugated steroid, the sulfate ester (S) and fatty acid esters (L). P + PS do not disappear from rat brain after combined adrenalectomy (adx) and castration (orx). PL does not serve a source of P after adx + orx. P is metabolized by several rat brain regions to progesterone and to PL. Brain microsomes contain the acyl-transferase which converts P to PL using endogenous substrates. Brain P and dehydroepiandrosterone (D) undergo a prominent circadian variation with their acrophases at the beginning of the dark span. The circadian variation of brain D persists after adx + orx. The monkey brain (Macaca fascicularis) also accumulates P and D. Adrenal suppression with dexamethasone for 4 days does not decrease the concentrations of brain P and 3rd ventricle CSFP and D. The concentrations of brain D are decreased to a much smaller extent than plasma D. D inhibits the aggressive behavior of castrated male mice exposed to lactating female intruders. This is not the case for DS or androst-5-ene-3 beta, 17 beta-diol. The D analog 3 beta-methyl-androst-5-en-17-one, which is not estrogenic and cannot be metabolized to testosterone or estradiol, is as active as D in inhibiting the aggressive behavior of castrated mice.     

Increase of brain tryptophan and stimulation of serotonin synthesis by salicylate

I ndirect evidence indicates that the rate-limiting step in the synthesis of brain 5-HT is the concentration of tryptophan in brain and not, as previously considered (G reen and S awyer , 1966), tryptophan hydroxylase. In fact this enzyme has a K_m for its substrate much higher than the concentration of tryptophan normally present in the mammalian brain (J equier , L ovenberg and S joerdsma , 1967; J equier , R obinson , L ovesberg and S joerdsma , 1969; M cgeer , P eters and M cgeer , 1968). Tryptophan is the only amino acid circulating in plasma which is highly bound to serum proteins (M cmenamy and O ncley , 1958). We have previously shown that the free fraction of serum tryptophan controls the concentration of brain tryptophan and, therefore, 5-HT synthesis as well (T agliamonte , B iggio and G essa , 1971d; G essa , B iggio and T agliamonte , 1972). Salicylate has been shown to displace tryptophan from its protein binding in plasma and to raise the free tryptophan concentration (M carthur and D awkins , 1969; S mith and L akatos , 1971). These considerations prompted us to study the effect of salicylate on tryptophan concentrations and 5-HT metabolism in brain.     

Brain transfer of a new neuromodulating ACTH analog, ebiratide, in rats.

The transfer of ebiratide into the brain was examined in rats. Its brain levels after intravenous administration (2-20 mg/kg), determined by radioimmunoassay, peaked at 5 min and declined almost in parallel with the plasma levels. Brain/plasma ratios (B/P) were constantly about 0.05 ml/g at all doses. Brain distribution study at 5 min after 125I-ebiratide at an effective dose (0.4 microgram/kg) revealed that unchanged ebiratide had larger B/P than the metabolites and region selectivity. The combined use with unlabeled ebiratide resulted in marked decreases in B/P, particularly in the hippocampus, suggesting a specific uptake of this peptide.     

Comparison of the effects of perfusion in determining brain penetration (brain-to-plasma ratios) of small molecules in rats

In the process of drug discovery, brain and plasma measurements of new chemical entities in rodents are of interest, particularly when the target receptors are in the brain. Brain-to-plasma ratios (B/P) obtained from a rodent pharmacokinetic assay are useful in helping determine which compounds are brain penetrant. The study reported here was performed to determine whether whole-body saline perfusion for complete blood removal was required to accurately measure brain tissue compound concentrations. Diazepam was used as a positive control since it is highly brain penetrant. Compound A was used as a negative control since it had known poor brain penetration. After intravenous dosing with either diazepam or compound A, rats were anesthetized and blood was collected, then the brain was removed following no perfusion or whole-body perfusion with saline. The analytes described (compound A, diazepam, and the internal standard) were recovered from plasma or brain homogenate by use of protein precipitation, and were subsequently analyzed by use of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The B/P values determined by use of LC-MS were not significantly different in perfused vs. non-perfused rats (P > or = 0.05). This approach (whole brain collected from non-perfused male rats) is an attractive alternative over brain penetration studies of perfused rats, since it has markedly reduced the technical time and potential for pain and distress required for generating B/P data due to elimination of the requirement for anesthesia and surgical preparation of animals.     

Changes in strand 6B and helix B during neuroserpin inhibition: Implication in severity of clinical phenotype

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.     

Inhibition of brain damage by edaravone, a free radical scavenger, can be monitored by plasma biomarkers that detect oxidative and astrocyte damage in patients with acute cerebral infarction

We assess the availability of plasma biomarkers to monitor the brain damage and the therapeutic efficacy of edaravone. The study consisted of 51 patients with ischemic cerebral infarcts. They were divided into 2 groups: GI (n = 24) had cortical lesions, and GII (n = 27) had lesions in the basal ganglia or brain stem. Edaravone was administered to 27 randomly selected patients (GIa, n = 13; GIIa, n = 14) and its efficacy was studied by comparing their plasma OxLDL, S-100B, and MnSOD levels to those in patients without edaravone (GIb, n = 11, GIIb, n = 13). Three days after the start of edaravone, plasma OxLDL was significantly lower in GIa than GIb patients (0.177 +/- 0.024 ng/microg apoB vs 0.219 +/- 0.026, P < 0.05). In GIIa patients, pre- and posttreatment plasma OxLDL was not significantly different (0.156 +/- 0.013 vs 0.152 +/- 0.020). In GIa patients, S-100B and MnSOD were significantly lower than in GIb patients (P < 0.05). The neurological condition at the time of discharge had recovered in GIa but not GIb patients. Ours is the first evidence to confirm the efficacy of edaravone by plasma biomarkers. In patients with cortical infarcts, edaravone reduced oxidative damage, thereby limiting the degree of brain damage.     

硫酸镁对脑源性肺损伤综合征的影响

目的探讨不同剂量的硫酸镁(MgSO4)对大鼠脑源性肺损伤后神经源性肺水肿、血浆炎性因子TNF-α及肺组织病理形态学变化的影响。方法将30只SD雄性大鼠按随机数字法分为假手术组(A组)、模型组(B组)及硫酸镁50mg/kg干预组(C组)、硫酸镁100mg/kg干预组(D组)、硫酸镁200mg/kg干预组(E组),每组6只。建立大鼠颅脑损伤模型后,硫酸镁干预组即刻按50mg/kg 25%MgSO4腹腔注射,C组注射1次、D组注射2次、E组注射4次,每8h注射1次。A组及B组的大鼠注射相同剂量生理盐水作对照,C组大鼠注射1次及D组大鼠注射2次MgSO4后给予注射相同剂量的生理盐水作对照,注射方法及间隔时间同E组。伤后48h测定大鼠肺组织含水量、血浆TNF-α浓度,肺组织常规HE染色,光镜观察肺组织病理形态学变化。结果大鼠颅脑创伤后肺组织含水量均高于假手术组,以C组最明显(P0.05),差异有统计学意义。B、C、D、E组大鼠之间肺组织含水量比较差异无统计学意义。B、C、D、E组大鼠TNF-α浓度均明显高于假手术组(P0.05),D组血浆TNF-α浓度明显低于B组(P0.05),E组血浆TNF-α浓度明显低于B组(P0.01),其他各组间差异无统计学意义。假手术组肺组织形态正常,肺血管无扩张,无炎症细胞浸润;B、C、D、E组与假手术组比较均可见终末支气管腔内充满炎症细胞,周围肺组织的肺泡腔内可见炎细胞浸润,肺血管扩张、充血。B、C、D、E组在炎症细胞浸润及肺毛细血管扩张方面无明显差异。结论脑外伤可导致脑源性肺损伤综合征,可导致神经源性肺水肿;硫酸镁可降低大鼠脑损伤后血浆TNF-α浓度,对肺水肿无明显影响。     

GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.

Recombinant GABAA (gamma-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of alpha-, beta-, and gamma 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Lüddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only alpha- and beta-subunit proteins have so far been detected. To identify the size and distribution of the gamma 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat gamma 2-subunit sequence. On Western blots, both anti-gamma 2-subunit antisera selectively labeled a 43-kDa protein. gamma 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the gamma 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the alpha 1-subunit, whereas bd17 recognizes both the beta 2- and beta 3-subunits (Ewert, M., Shivers, B. D., Lüddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated approximately 90% of the GABAA receptors, the gamma 2-subunit is frequently associated with the alpha 1-subunit and the beta 2- and/or beta 3-subunit in vivo.     

Perturbations in factors that modulate osteoblast functions in vitamin B6 deficiency

It was hypothesized that the widespread structural defect of collagen in connective tissue of vitamin B6 deficient-animals and the consequent alteration in bone biomechanical properties cause an additional stress to their inflamed swollen tibiotarsometatarsal joints. The present study showed a 32% elevation (P < 0.02) in mean plasma free cortisol concentration. Vitamin D metabolism was impaired but without changing plasma calcium homeostasis and bone mineral content. Mean plasma calcitriol [1,25(OH)2D] concentration was significantly reduced (P < 0.001). Because plasma calcidiol concentration did not change, we speculated that either renal 25-hydroxycalciferol-1alpha-hydroxylase activity was reduced or 1,25(OH)2D turnover was increased. Plasma osteocalcin, an index of osteoblast function related to bone formation, was significantly decreased (P < 0.05). This adverse effect on osteoblasts was consistent with the reduction of bone specific alkaline phosphatase activity (another index of bone formation) found in a previous study. The excess of cortisol may have impaired these bone cells functions directly and (or) indirectly via the decline in calcitriol synthesis. Plasma hydroxyproline concentrations in B6-deficient animals were found to be significantly reduced (P < 0.001), suggesting that cortisol in excess had also a suppressive effect on another hydroxylase, namely tissue (mainly bone and liver) prolyl hydroxylase. The bone uncoupling (in formation and resorption) associated with vitamin B6 deficiency seems to be due to secondary hypercortisolism and (or) another unknown factors but not related to a change in bone modulators such as IGF-1 and eicosanoids.     

Restricted Transport of Vitamin D and A Derivatives Through the Rat Blood-Brain Barrier

The present studies measure the transport of retinol, retinoic acid, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and 25-hydroxyvitamin D3 [25-(OH)D3] through the rat brain capillary endothelial wall, i.e., the blood-brain barrier (BBB). The vitamin A and D derivatives bind both to albumin and to specific high-affinity binding proteins in plasma. In the presence of physiologic concentrations of plasma proteins, the extraction by brain of all four compounds was 5% or less.     

Epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (P=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (P=0.006). In both females and males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (P=0.026 and P=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology.     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

Influence of treatment with 3′-deoxyadenosine associated deoxycoformycin on hematological parameters and activity of adenosine deaminase in infected mice with Trypanosoma evansi

This study aimed to verify the effect of 3′-deoxyadenosine and deoxycoformycin on hematologic parameters and adenosine deaminase (ADA) activity in plasma and brain of mice infected with Trypanosoma evansi. Seventy animals were divided into seven groups, which were divided into two subgroups each for sampling on days 4 and 8 post-infection (PI). The groups were composed of three uninfected groups (A–C), namely, not-treated (A), treated with 3′-deoxyadenosine (B), and treated with deoxycoformycin (C) and four infected groups, mice with T. evansi (D–G), namely, not-treated (D), treated with 3′-deoxyadenosine (E), treated with deoxycoformycin (F), and treated with a combination 3′-deoxyadenosine and deoxycoformycin (G). Hematological parameters and ADA activity were evaluated in plasma and brain. Animals in groups B and C exhibited a reduction in the levels of plasma total protein compared group A. Animals in groups D and F showed changes in the hematological parameters. The ADA activity significantly reduced in the animals of groups C, D, F and G. Mice in the group E presented increased ADA activity in plasma. Therefore, we conclude that the treatment interferes significantly in the hematologic parameters in mice infected with T. evansi. On the other hand, when the ADA inhibitor was used we observed a significant decrease in the values of hematocrit, total erythrocytes, and hemoglobin concentration. The deoxycoformycin was able to inhibit the ADA activity of parasite thus it may be one of the mechanisms of efficacy of this treatment.     

The synthesis of sphingomyelin in the Morris hepatomas 7777 and 5123D is restricted to the plasma membrane

The site of sphingomyelin synthesis in the rapidly growing Morris hepatomas 7777 and 5123D was determined by incubating plasma membrane, mitochondrial and microsomal membrane fractions with vesicles of phosphatidyl[methyl-14C]choline in the presence of phosphatidylcholine transfer protein. In agreement with a previous study on rat liver (Voelker, D.R. and Kennedy, E.P., 1982, Biochemistry 21, 2753-2759) we have demonstrated that sphingomyelin synthesis in these hepatomas is restricted to the plasma membrane. The greatly elevated sphingomyelin content of mitochondria and microsomes (Hostetler, K.Y., Zenner, B.D. and Morris, H.P., 1979, Cancer Res. 39, 2978-2983) suggests that rapidly growing hepatomas, in contrast to liver, have an effective mechanism of intracellular sphingomyelin transfer.     

Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeability

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..     

Taurine in plasma and CSF: a study in healthy male volunteers

In order to explore the interrelationship between plasma and cerebrospinal fluid taurine concentrations, three consecutive 6-ml fractions of cerebrospinal fluid were drawn from 30 healthy male volunteers in the early morning after 8 h in the fasting condition. Repeated plasma samples were drawn over 24 h the day before lumbar puncture. Taurine in plasma and cerebrospinal fluid was determined by high performance liquid chromatography. The subjects were categorized as extensive or poor metabolizers with respect to the cytochrome P450 2D6 genotype. The taurine cerebrospinal fluid/plasma ratio at 8 a.m. was negatively influenced by the plasma taurine concentration at 4 p.m. the previous day. It was also negatively influenced by body mass index and positively by the intraspinal pressure. Three poor metabolizers of cytochrome P450 2D6 had higher plasma taurine areas under the curve than 27 extensive metabolizers. Hypothetically, cytochrome P450 2D6 influences the transport of taurine across the blood–brain barrier.     

Distribution of alpha1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

Distribution of alpha(1)-adrenoceptor (alpha(1)AR) subtype (alpha(1A), alpha(1B), alpha(1D)) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive alpha(1A)AR and alpha(1D)AR in brain and heart and of alpha(1B)AR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of alpha(1A)AR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of alpha(1B)AR was higher in brain and heart and that of alpha(1D)AR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of alpha(1B)AR increased in the heart but decreased in the liver; alpha(1A)AR and alpha(1D)AR in liver, kidney, and heart were not affected by age. It is concluded that alpha(1)AR subtypes are widely expressed in different neonatal and adult rat tissues.     

Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes

Posttranslational modifications and intracellular transport of the D2- cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.     

The combined effect of pomegranate extract and tangeretin on the DMBA-induced breast cancer model

The aim of this study was to investigate the protective effects of pomegranate extract and tangeretin alone or in combination in DMBA-induced rat breast cancer model.A total of 68 female rats were randomly divided into 8 groups. The first 4 groups were designed as controls for cancer and treatment groups, and the control groups were composed of only control (C), Pomegranate (P), Tangeretin (T), and Pomegranate+Tangeretin (P+T) groups. The other four groups were designed as cancer and treatment groups and were composed of DMBA (D) and DMBA+Pomegranate (D+P), DMBA+Tangeretin (D+T), DMBA+Pomegranate+Tangeretin (D+P+T) groups. Tumor markers and angiogenesis parameters were studied from plasma samples obtained from rats. Histopathological, immunohistochemical, and TUNEL analyses and expressions of proteins affecting apoptosis and cell cycle were determined in breast tissue samples.In the DMBA group, plasma CA15-3, CEA, VEGF, MMP-9, and NF-κB levels were significantly increased compared to the controls, but significant decreases were observed in these parameters except MMP-9 in the treatment groups. It was observed that p53 and Bax expressions significantly increased in both D+P and D+P+T groups compared to the DMBA group, and these findings were supported by Tunel and immunohistochemical findings. Cyclin D1 expressions were found to be significantly decreased only in the D+T group and supported by TUNEL and immunohistochemical findings. Immunohistochemical ER-α and Ki-67 immune reactivities were significantly decreased in all treatment groups compared to the DMBA group.Our results showed that combined application of pomegranate extract and tangeretin may be more beneficial in preventing breast cancer development.