[Equol and its enantiomers inhibited urethane-induced lung cancer in mice].

OBJECTIVE: To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice. METHODS: A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison. RESULTS: A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups. CONCLUSION: Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.

Equol and its enantiomers inhibited urethane-induced lung cancer in mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice.@*METHODS@#A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison.@*RESULTS@#A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups.@*CONCLUSION@#Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.

More evidence for a long-latency mismatch response in urethane-anaesthetised mice.

Long-latency mismatch responses to oddball stimuli have recently been observed from anaesthetised rodents. This electrophysiological activity is viewed through 200 to 700 ms post-stimulus; a window that is typically obstructed from analysis by the response to subsequent stimuli in the auditory paradigm. A novel difference waveform computation using two adjoining evoked responses has enabled visualisation of this activity over a longer window than previously available. In the present study, this technique was retroactively applied to data from 13 urethane-anaesthetised mice. Oddball paradigm waveforms were compared with those of a many-standards control sequence, confirming that oddball stimuli evoked long-latency potentials that did not arise from standard or control stimuli. Statistical tests were performed to identify regions of significant difference. Oddball-induced mismatch responses were found to display significantly greater long-latency potentials than identical stimuli presented in an equal-probability context. As such, it may be concluded that long-latency potentials were evoked by the oddball condition. How this feature of the anaesthetised rodent mismatch response relates to human mismatch negativity is unclear, although it may be tentatively linked to the human P3a component, which emerges downstream from mismatch negativity under certain conditions. These results demonstrate that the time dynamics of mismatch responses from anaesthetised rodents are more extensive than previously considered.

Novel therapeutic regimens for urethane-induced early lung cancer in rats: Combined cisplatin nanoparticles with vitamin-D3.

Lung cancer remains incurable; therefore, novel therapeutical approaches are of great demand. This study was designed to investigate the effectiveness of cisplatin nanoparticles combined with vitamin-D3 on urethane-induced early lung cancer in rats and to clarify the underlying signaling mechanisms. Early lung cancer was induced in male Wistar rats by urethane. Rats were divided into six groups: I-control, II-cancer untreated, III-cancer + free cisplatin, IV-cancer + cisplatin nanoparticles, V-cancer + free cisplatin + vitamin-D3 , VI-cancer + cisplatin nanoparticles + vitamin-D3 . Inflammation, proliferation, and apoptosis were evaluated together with the levels of tumor marker CK-19 along with histological assessment. Treatment of lung cancer with either free or nanoparticles of cisplatin alone demonstrated significant suppression in the expression of inflammatory, anti-apoptotic and tumor markers compared to rats with lung cancer. Moreover, vitamin-D3 supplementation with either cisplatin forms lead to a further decrease of all markers, markedly with cisplatin nanoparticles. The present study shows the synergistic effect of cisplatin-nanoparticles combined with vitamin-D3 as a new therapy regimen against lung cancer. Further studies with larger sample sizes and longer duration are needed to confirm these results.

New function of polysaccharide from Rubus chingii Hu: protective effect against ethyl carbamate induced cytotoxicity.

BACKGROUND: Rubus chingii Hu is a widely cultivated fruit in China and has declared multiple bioactivities including antioxidative activity. Ethyl carbamate (EC), mostly found in fermented food and alcoholic beverages, is a recognized human carcinogen, and researchers have proposed the correlation between oxidative stress and its toxicity. This study acquired the polysaccharide from R. chingii (RP) and explored its effect on EC-induced cytotoxicity using Caco-2 cells as the cell model. RESULTS: Results showed that RP exhibited protection against EC-induced toxicity by repairing redox imbalance as indicative of mitigated mitochondrial membrane potential collapse, attenuated reactive oxygen species overproduction, and impeded glutathione depletion. Moreover, the structural features of RP were characterized and revealed that it was mainly constituted by galacturonic acid and arabinose, with an average molecular weight of 7.039 × 105 g mol-1 . CONCLUSION: Overall, our results provided a new approach dealing with the toxicity caused by EC from the perspective of oxidative stress and described a new potential healthy value of R. chingii Hu, which could contribute to the development of a promising dietary supplement and functional food. © 2020 Society of Chemical Industry.

Evodiamine suppresses Notch3 signaling in lung tumorigenesis via direct binding to γ-secretases.

BACKGROUND: Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. PURPOSE: We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. METHODS: Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. RESULTS: EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. CONCLUSION: This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.

A synergistic approach for management of lung carcinoma through folic acid functionalized co-therapy of capsaicin and gefitinib nanoparticles: Enhanced apoptosis and metalloproteinase-9 down-regulation.

BACKGROUND: Lung cancer is one of the most lethal cancers and lacks effective treatment strategy. Therapeutic efficacy can be improved through active targeting approach utilizing surface engineered nanoparticles (NPs) for cancer therapy. PURPOSE: The present study envisioned development of Folic acid (FA) functionalized NPs for co-administration of gefitinib (Gnb) and capsaicin (Cap) respectively to enhance the therapeutic outcome by disabling the barriers related to tumors extracellular matrix. RESEARCH METHODS AND PROCEDURE: The FA conjugated Gnb/Cap polymeric (PLGA-PEG) NPs were prepared using oil in water emulsion technique and methodically developed using Quality by Design (QbD) concept employing central composite design. The developed formulations were subjected to various in vitro (A549 cell lines) and in vivo evaluations in urethane-induced lung cancer. RESULTS: The modified NPs displayed particle sizes of 217.0 ±â€¯3.2 nm and 213.0 ±â€¯5.2 nm and drug release of 85.65 ±â€¯3.21% and 81.43 ±â€¯4.32% for Gnb and Cap respectively. Higher cellular uptake and lower cell viability in A549 cell line was displayed by functionalized NPs compared to free drug. Co administration of Gnb and Cap NPs displayed significant targeting potential, reduction in tumor volume while restoring the biochemical parameters viz., SOD, catalase, TBARS and protein carbonyl, towards normal levels when compared with toxic group. Significant down regulation was observed for anti-apoptotic proteins (MMP-9) and up regulation of pro-apoptotic proteins (caspase-3, caspase-9 and MMP-9) with co-therapy of Gnb and Cap NPs, when compared with individual therapy through Gnb/Cap. CONCLUSION: Potentiation of the action of Gnb when co administered with Cap NPs can be a promising breakthrough for developing safe, effective and targeted delivery for lung carcinoma therapy.

Liquid crystalline assembly for potential combinatorial chemo-herbal drug delivery to lung cancer cells.

BACKGROUND: Lung cancer is the most common cancer and the leading cause of total deaths worldwide. Its classified into two major types including non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) based on the origin of abnormal lung cells as well as the smoking status of the patient. NSCLC is the most common and aggressive type of lung cancer representing 80%-85% of all cases. PURPOSE: The aim of the study was to present lyotropic liquid crystalline nanoparticles (LCNPs) as promising carriers for co-delivery of the chemotherapeutic agent, pemetrexed (PMX) and the herbal drug, resveratrol (RSV) for effective lung cancer management. METHODS: The proposed PMX-RSV-LCNPs were prepared by hydrotrope method. Hydrophobic ion pairing with cetyl trimethyl ammonium bromide (CTAB) was implemented to increase the encapsulation efficiency of the hydrophilic PMX up to 95%±3.01%. RESULTS: The tailored PMX-RSV-LCNPs exhibited a particle size of 173±0.26 nm and biphasic release pattern with a relatively initial burst release within first 3-4 hour followed by sustained release up to 24 hours. Moreover, PMX-RSV-LCNPs manifested superior concentration and time dependent cytotoxicity profile against A549 lung cancer cells with IC50 4.0628 µg/mL. Besides, the enhanced cellular uptake profile based on bioadhesive properties of glyceryl monoolein (GMO) as well as energy independent (cholesterol dependent) pattern. In-vivo evaluations against urethane induced lung cancer bearing mice demonstrated the potentiality of PMX-RSV-LCNPs in tumor growth inhibition via inhibition of angiogenesis and induction of apoptosis. The results were supported by histopathological analysis and immunohistochemical Ki67 staining. Moreover, PMX-RSV-LCNPs displayed a promising safety profile via attenuating nephro- and hepatotoxicity. CONCLUSION: PMX-RSV-LCNPs elaborated in the current study hold a great promise for lung cancer treatment.

Comparison of the protective effect of black and white mulberry against ethyl carbamate-induced cytotoxicity and oxidative damage.

Increasing evidence indicates that crude extracts derived from mulberry confer protection against oxidative stress. However, the antioxidant capacity of mulberry extract among different cultivars remains elusive. The main objective of the present study was therefore to investigate the effect of black mulberry extract (BMB) and white mulberry extract (WMB) on ethyl carbamate (EC)-induced cytotoxicity and oxidative stress. This study showed that the contents of total phenolics, total flavonoids, total procyanidins, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and pelargonidin-3-O-glucoside of BMB were higher than that of WMB. Moreover, our results showed that phenolics-abundant BMB was stronger than WMB in scavenging ABTS and DPPH free radicals. BMB was more effective in ameliorating EC-induced cytotoxicity by inhibiting excessive ROS generation, suppressing mitochondrial dysfunction and increasing GSH concentration in HepG2 cells than WMB. Taken together, our study revealed that BMB afforded better protection against EC-caused cytotoxicity than WMB.

The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model.

Prostacyclin (prostaglandin I2, PGI2) overproduction in FVB/N mice prevents the formation of carcinogen and tobacco smoke-induced adenomas, and administration of the oral prostacyclin analogue iloprost to wild-type mice also prevented carcinogen-induced mouse lung adenoma formation. Former smokers taking oral iloprost showed improved bronchial dysplasia histology compared with placebo. Next-generation oral prostacyclin analogues, like treprostinil, were developed for the treatment of pulmonary arterial hypertension (PAH). On the basis of our prior studies with iloprost, we performed preclinical studies examining the ability of treprostinil to chemoprevent urethane-induced murine lung adenocarcinoma. We determined the MTD in chow (prior studies had delivered treprostinil by gavage), and this dose produced serum levels in the experimental animals similar to those found in PAH patients treated with treprostinil. We then examined the chemopreventive efficacy of treprostinil exposure initiated both before (1 week) and after (6 weeks) urethane exposure to better model chemoprevention studies conducted in former smokers. Neither of these dosing strategies prevented murine lung cancer; however, we did detect changes in pulmonary inflammatory cell infiltrate and expression of CXCR4 (a chemokine receptor previously shown to increase in response to treprostinil exposure) in tumor-bearing, treprostinil-treated animals, indicating that the drug was bioavailable. One potential explanation stems from iloprost and treprostinil differentially activating cell surface prostaglandin receptors and intracellular peroxisome proliferator-activated receptors. When murine lung tumor cells were treated with treprostinil, their proliferation rate increased; in contrast, iloprost had no effect on proliferation. Future investigations comparing these two agents will provide insight into iloprost's chemopreventive mechanisms. Cancer Prev Res; 10(11); 671-9. ©2017 AACR.

Blackberry subjected to in vitro gastrointestinal digestion affords protection against Ethyl Carbamate-induced cytotoxicity.

Ethyl Carbamate (EC) was detected in many fermented foods. Previous studies indicated that frequent exposure to ethyl carbamate may increase the risk to suffer from cancers. Blackberry is rich in polyphenols and possesses potent antioxidant activity. This study aims to investigate the protective effect of blackberry homogenates produced before (BH) and after in vitro simulated gastrointestinal digestion (BD) on EC-induced toxicity in Caco-2 cells. Our results showed that blackberry homogenates after digestion (BD) was more effective than that before digestion (BH) in ameliorating EC-induced toxicity in Caco-2 cells. Further investigation revealed that BD remarkably attenuated EC-induced toxicity through restoring mitochondrial function, inhibiting glutathione depletion and decreasing overproduction of intracellular reactive oxygen species. Additionally, LC-MS result implied that the better protective capacity of BD may be related to the increased content of two anthocyanins (cyanidin-3-glucoside and cyanidin-3-dioxalyglucoside). Overall, the present study may give implication to prevent EC-induced health problem.

Chemopreventive effect of a novel nutrient mixture on lung tumorigenesis induced by urethane in male A/J mice.

AIMS AND BACKGROUND: Lung cancer, a leading cause of cancer death, is associated with exposure to inhalation carcinogens, most commonly those found in tobacco smoke. We investigated the in vivo effect of dietary supplementation with a nutrient mixture containing lysine, proline, arginine, ascorbic acid, green tea extract, N-acetyl cysteine, selenium, copper and manganese on the development of urethane-induced lung tumors in male A/J mice. METHODS: After one week of isolation, seven-week-old male A/J mice (n = 25) weighing 17-19 g were randomly divided into three groups: group A (n = 5), group B (n = 10), and group C (n = 10). Mice in groups B and C were each given a single intraperitoneal injection of urethane (1 mg/g body weight) in saline, whereas group A mice received an injection of saline alone. Groups A and B were fed a regular diet, whereas group C was fed the same diet supplemented with 0.5% nutrient mixture. After 20 weeks, mice were sacrificed, lungs were excised and weighed, and tumors were counted and processed for histology. RESULTS: Urethane-challenged mice developed tumors. However, the mean number of tumors and the mean lung weights in the mice on the supplemented diet were significantly reduced, by 49% (P < 0.0001) and 18% (P = 0.0025), respectively, compared to mice on the control diet. We observed neither significant differences in body weight gains nor in diet consumption among the mice. Pulmonary lesions were morphologically similar for both the groups (adenomas), but lesions were smaller in the test group. CONCLUSIONS: The results suggest that nutrient mixture has inhibitory potential on the development of mouse lung tumors induced by urethane.

Inhibition of vinyl carbamate-induced mutagenicity and clastogenicity by the garlic constituent diallyl sulfone in F1 (Big Blue x A/J) transgenic mice.

Vinyl carbamate (VC) is a metabolite of ethyl carbamate (EC), a naturally occurring compound found in fermented foods and alcoholic beverages. CYP2E1 mediates the sequential oxidation of EC to VC and subsequently to the vinyl carbamate epoxide, which is believed to be the ultimate carcinogenic species. Here, we have tested the hypothesis that bioactivation of VC by CYP2E1 plays a central role in the development of its mutagenicity and clastogenicity, and further that inhibition of CYP2E1 by diallyl sulfone (DASO(2)) leads to diminution in their incidences. DASO(2) is a garlic constituent that is oxidized by CYP2E1, leading to inactivation of this P450. F(1) (Big Blue x A/J) transgenic mice harboring the lambda cII gene were used for in vivo identification and quantitation of mutations in the lung and small intestine. Mice were pre-treated with DASO(2) (12.5-200 mg/kg, p.o.), treated 2 h later with VC (60 mg/kg, i.p.) and were killed 4 weeks later. Our results showed that pre-treatment of mice with DASO(2) at doses of 50-200 mg/kg significantly decreased the VC-induced mutant frequencies (MFs) by 50-70%. In the small intestine, pre-treatment with 200 mg/kg of DASO(2) decreased the MF by approximately 40%. Clastogenicity, as assessed by the frequency of micronucleated reticulocytes, was significantly decreased (33-44%) by pre-treatment with DASO(2) (50-200 mg/kg). These results demonstrated that bioactivation of VC by CYP2E1 plays a valid role in the development of mutagenicity and clastogenicity, and further that inhibition of this pathway by DASO(2) produces a protective effect.

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents.

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.

Interactions in the tumor-promoting activity of carbon tetrachloride, trichloroacetate, and dichloroacetate in the liver of male B6C3F1 mice.

Interactions between carcinogens in mixtures found in the environment have been a concern for several decades. In the present study, male B6C3F1 mice were used to study the responses to mixtures of dichloroacetate (DCA), trichloroacetate (TCA), and carbon tetrachloride (CT). TCA produces liver tumors in mice with the phenotypic characteristics common to peroxisome proliferators. DCA increases the growth of liver tumors with a phenotype that is distinct in several respects from those produced by TCA. These chemicals are effective as carcinogens at doses that do not produce cytotoxicity. Thus, they encourage clonal expansion of initiated cells through subtle, selective mechanisms. CT is well known for its ability to promote the growth of liver tumors through cytotoxicity that produces a generalized growth stimulus in the liver that is reflected in a reparative hyperplasia. Thus, CT is relatively non-specific in its promotion of initiated cells within the liver. The objective of this study was to determine how the differing modes of action of these chemicals might interact when given as mixed exposures. The hypothesis was that the effects of two selective promoters would not be more than additive. On the other hand, CT would be selective only to cells not sensitive to its effects as a cytotoxin. Thus, it was hypothesized that neither DCA nor TCA would add significantly to the effects produced by CT. Mice were initiated by vinyl carbamate (VC), and then promoted by DCA, TCA, CT, or the pair-wised combinations of the three compounds. The effect of each treatment or treatment combination on tumor number per animal and mean tumor volume was assessed in each animal. Dose-related increases in mean tumor volume were observed with 20 and 50mg/kg CT, but each produced equal numbers of tumors at 36 weeks. As the dose of CT was increased to >/=100mg/kg substantial increases in the number of tumors per animal were observed, but the mean tumor size decreased. This finding suggests that initiation occurs as doses of CT increase to >/=100mg/kg, perhaps as a result of the inflammatory response that is known to occur with high doses of CT. When administered alone in the drinking water at 0.1, 0.5 and 2g/l, DCA increased both tumor number and tumor size in a dose-related manner. With TCA treatment at 2g/l in drinking water a maximum tumor number was reached by 24 weeks and was maintained until 36 weeks of treatment. DCA treatment did not produce a plateau in tumor number within the experimental period, but the numbers observed at the end of the experimental period were similar to TCA and doses of 50mg/kg CT. The tumor numbers observed at the end of the experiment are consistent with the assumption that the administered dose of the tumor initiator, vinyl carbamate, was the major determinant of tumor number and that treatments with CT, DCA, and TCA primarily affected tumor size. The results with mixtures of these compounds were consistent with the basic hypotheses that the responses to tumor promoters with differing mechanisms are limited to additivity at low effective doses. More complex, mutually inhibitory activity was more often observed between the three compounds. At 24 weeks, DCA produced a decrease in tumor numbers promoted by TCA, but the numbers were not different from TCA alone at 36 weeks. The reason for this result became apparent at 36 weeks of treatment where a dose-related decrease in the size of tumors promoted by TCA resulted from DCA co-administration. On the other hand, the low dose of TCA (0.1g/l) decreased the number of tumors produced by a high dose of DCA (2g/l), but higher doses of TCA (2g/l) produced the same number as observed with DCA alone. DCA inhibited the growth rate of CT-induced tumors (CT dose = 50mg/kg). TCA substantially increased the numbers of tumors observed at early time points when combined with CT, but this was not observed at 36 weeks. The lack of an effect at 36 weeks was attributable to the fact that more than 90% of the livers consisted of tumors and the earlier effect was masked by coalescence of tumors. Thus, the ability of TCA to significantly increase tumor numbers in CT-treated mice was probably real and contrary to our original hypothesis that CT was non-specific in its effects on initiated cells. It is probable that the interaction between CT and TCA is explained through stimulation of the growth of cells with differing phenotypes. These data suggest that the outcome of interactions between the mechanisms of tumor promotion vary based on the characteristics of the initiated cells. The interactions may result in additive or inhibitory effects, but no significant evidence of synergy was observed.

Antimutagenic properties of bell and black peppers.

The wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster was used to study the modulating action of bell pepper (Capsicum annuum) and black pepper (Piper nigrum) in combination with the alkylating agent methyl methanesulfonate (MMS) and the promutagen agent ethyl carbamate (EC). Larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 [flr(3)] were fed genotoxins alone or in combination with each of the two spices. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Our results showed that bell pepper was effective in reducing the mutational events induced by EC and MMS and black pepper was only effective against EC. Pretreatment of 2-day-old larvae with the spices for 24 h followed by a treatment with EC and MMS was only effective in reducing mutations induced by EC. Suppression of metabolic activation or interaction with the active groups of mutagens could be mechanisms by which the spices exert their antimutagenic action.

Delayed-type hypersensitivity and humoral immunity modulation by dietary lipids in a murine model of pulmonary tumorigenesis induced by urethan.

Mice fed on semisynthetic formulas containing 15% of corn oil (CO), cod fish liver oil (FO), oleic acid (O) or a mixture of 46% of palmitic and 50% of stearic acids (PS) were treated with urethan during 18 weeks for lung tumor induction. Delayed-type hypersensitivity (DTH) assay, hemagglutination assay and the amount of lung nodes (alveolar adenocarcinomas) were recorded. Results showed significantly greater DTH in CO and FO with respect to O and PS feeding mice; the two last ones induced an essential fatty acid (EFA) deficiency (EFAD). In the O lot there was a non-significant diminution of the humoral response. EFAD animals exhibited a tendency to increase number of lung nodes in relation to CO and FO lots. Splenomegalia was recorded in FO lot. Confront between spleen weight and DTH showed a 72% correlation, suggesting an increase in cellular immunity as increasing unsaturation. It may be concluded that in this suitable model of tumorigenesis the manipulation of dietary lipids may be a strategy to modify the immune system response.

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations.

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Nonspecific stimulation of the maternal immune system. II. Effects on gene expression in the fetus.

BACKGROUND: Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS: Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freund's complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS: Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS: These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.

Chemoprevention of vinyl carbamate-induced lung tumors in strain A mice.

The ability of potential chemopreventive agents to prevent vinyl carbamate-induced lung tumors was determined in 2 different experiments. Female strain A mice administered intraperitoneally either a single injection of 60 mg/kg vinyl carbamate that induced 24.0 +/- 1.72 tumors/mouse at 24 weeks or 2 injections of 16 mg/kg vinyl carbamate each (32 mg/kg total dose) that induced 43.2 +/- 3.2 tumors/mouse at 20 weeks. Lung carcinomas were found as early as 16 weeks. Dexamethasone and piroxicam provided in the diet were found to significantly inhibit lung tumors induced by 60 mg/kg vinyl carbamate at 24 weeks whereas myo-inositol also provided in the diet, did not significantly inhibit tumor formation. In animals given 6 16-mg/kg doses of vinyl carbamate, tumor multiplicity was reduced roughly 25% by alpha-difluoromethylornithine and green tea and reduced 50% by dexamethasone and piroxicam. Combinations of these agents were also tested using a total dose of 32 mg/kg of vinyl carbamate. Although alpha-difluoromethylornithine and green tea did not result in a significant inhibition of lung tumor formation if used alone, the combination of alpha-difluoromethylornithine and green tea resulted in a significant reduction of tumor multiplicity. The combinations of alpha-difluoromethylornithine or green tea with either dexamethasone or piroxicam or the combination of dexamethasone and piroxicam did not decrease tumor multiplicity greater than achieved by dexamethasone and piroxicam alone. In summary, selected chemopreventive agents previously shown to inhibit lung tumors by other chemical carcinogens also inhibited vinyl carbamate-induced lung tumors.