Modifying effect of propolis on dimethylhydrazine-induced DNA damage but not colonic aberrant crypt foci in rats

Propolis is a honeybee product with several biological and therapeutic properties, including antimutagenic and anticarcinogenic activities. The effects of an aqueous extract of propolis (AEP) were evaluated on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and DNA damage in the colon of male Wistar rats by the ACF and Comet assays, respectively. AEP was administered orally at 0.01%, 0.03%, 0.1%, and 0.3% in the drinking water, which resulted in doses of approximately 12, 34, 108, and 336 mg/kg body weight/day. Animals were also given a single subcutaneous injection of 40 mg/kg DMH and sacrificed 4 hr later for evaluating DNA damage, or 4 doses of 40 mg/kg DMH, administered 2 doses/week for 2 weeks, and sacrificed 12 weeks after the last injection for evaluating ACF development in the distal colon. Administration of AEP either simultaneously with or after the DMH treatment resulted in no statistically significant reduction of ACF. In contrast, 0.01%, 0.03%, and 0.3% AEP, given simultaneously with DMH, reduced DNA damage induction in the mid and distal colon. However, 0.3% AEP alone increased DNA damage in the colon. In conclusion, AEP had no effect on the formation of DMH-induced ACF in rat colon, but it modulated DMH-induced DNA damage in colon cells. Further investigations are recommended in order to establish the conditions under which propolis produces either protective or deleterious effects.     

Ursolic acid and oleanolic acid suppress preneoplastic lesions induced by 1,2-dimethylhydrazine in rat colon

Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenoid compounds found in plants used in the human diet and in medicinal herbs, in the form of aglycones or as the free acid. These compounds are known for their hepatoprotective, anti-inflammatory, antimicrobial, hypoglycemic, antimutagenic, antioxidant, and antifertility activities. In the present study, we evaluated the effects of UA and OA on the formation of 1,2-dimethyl-hydrazine (DMH)-induced aberrant crypt foci (ACF) in the colon of the male Wistar rat. The animals received subcutaneous (sc) injections of DMH (40 mg/kg body weight) twice a week for two weeks to induce ACF. UA, OA and a mixture of UA and OA were administered to the rats five times a week for four weeks by gavage at doses of 25 mg/kg body weight/day each, during and after DMH treatment. All animals were sacrificed in week 5 for the evaluation of ACF. The results showed a significant reduction in the frequency of ACF in the group treated with the triterpenoid compounds plus DMH when compared to those treated with DMH alone, suggesting that UA and OA suppress the formation of ACF and have a protective effect against colon carcinogenesis.     

A new medium-term rat colorectal bioassay applying neoplastic lesions as end points for detection of carcinogenesis modifiers effects with weak or controversial modifiers

We have established a two-stage, medium-term rat colorectal carcinogenesis model featuring induction of neoplastic lesions within ten weeks. In the present study, we examined the ability of this model to detect weak modifiers. F344 male rats were given three subcutaneous (sc) injections of 1,2-dimethyl-hydrazine (DMH, 40 mg/kg b.w.) in one week followed by drinking water containing 1% dextran sodium sulfate (DSS) for a second week. One week after this regimen, basal diet alone, or diets containing 10% perilla oil, 10% corn oil, 10% dextrin, or 0.1% indole-3-carbinol (I3C) were supplied. The perilla oil and corn oil groups did not show significant differences in the numbers of aberrant crypt foci (ACF) and incidences or multiplicity of proliferative lesions as compared to the controls at either time point. In the dextrin group, the total number of ACF at week ten was significantly increased. With I3C, the total number of ACF and incidence and multiplicities of adenocarcinomas at week ten and the incidence of invasive tumors at week twenty were significantly increased. These data essentially correspond with earlier reported results, except in the vegetable oil cases. Thus, the system is suitable for detection of colorectal carcinogenesis modifiers with advantages over previous models using ACF alone as end points.     

Modulatory effect of troxerutin on biotransforming enzymes and preneoplasic lesions induced by 1,2-dimethylhydrazine in rat colon carcinogenesis

Colon cancer is the third most global oncologic problem faced by medical fraternity. Troxerutin, a flavonoid present in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and biological activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizing enzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydrazine (DMH) induced experimental rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control. Group 2 received troxerutin (50 mg/kg b.w., p.o. every day) for 16 weeks. Groups 3–6 received subcutaneous injections of DMH (20 mg/kg b.w.) once a week, for the first four weeks. In addition, groups 4–6 received different doses of troxerutin (12.5, 25, 50 mg/kg b.w., p.o. every day respectively) along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I enzymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liver and colonic mucosa of control and experimental rats. Furthermore, the activities of fecal and colonic mucosal bacterial enzymes, such as β-glucronidase, β-glucosidase, β-galactosidase and mucinase were found to be significantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin to DMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and the pathological changes were significantly reversed, the effect being more pronounced when troxerutin was supplemented at the dose of 25 mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agent against the formation of preneoplastic lesions in a rat model of colon carcinogenesis.     

Chemopreventive effects of vanadium toward 1,2-dimethylhydrazine-induced genotoxicity and preneoplastic lesions in rat colon

In the present study, we have evaluated the antitumor effects of vanadium by monitoring DNA damage and chromosomal aberrations (CAs) during the early preneoplastic stage of 1,2-dimethylhydrazine (1,2-DMH)-induced colon cancer in male rats. Treatment with 20 mg/kg 1,2-DMH for 6 weeks resulted in the formation of aberrant crypt foci (ACF), a putative preneoplastic lesion associated with colon cancer development, while cotreatment with ammonium monovanadate (0.5 ppm in the drinking water) reduced ACF formation by 50% (P < 0.001). The 6-week treatment with 1,2-DMH also resulted in significantly higher levels of DNA damage in rat colon as measured by the Comet assay (higher mean values for length-to-width ratios (L:W) of DNA mass (P < 0.01) and mean frequencies of cells with comets (P < 0.001)). The vanadium cotreatment reduced DNA damage in colon cells by 32% (P < 0.02 and P < 0.001 for L:W and tailed cells, respectively). 1,2-DMH treatment also produced a 10-fold increase in the frequency of CAs in rat colon (P < 0.001), while cotreatment with vanadium resulted in a reduction in CAs after 2, 4, and 6 weeks of 1,2-DMH exposure (P < 0.01). Analysis of antioxidant defense enzyme activity in colonic mucosa indicated that glutathione reductase and catalase activities were increased in 1,2-DMH-treated rats; cotreatment with vanadium reduced these activities when compared to the carcinogen control (P < 0.001 and P < 0.02). These results demonstrate that the early protective effect of vanadium in chemically induced rat colon carcinogenesis may be mediated by a reduction of carcinogen-induced DNA damage.     

Immunomodulatory Effects of Withania Somnifera on Azoxymethane Induced Experimental Colon Cancer in Mice

The efficacy of Withania somnifera on immunomodulation was tested in experimental azoxymethane induced colon cancer in mice. Azoxymethane is a potential carcinogen to induce the colon cancer in Swiss albino mice. Azoxymethane 15 mg/kg body weight was injected intraperitoneally once a week for 28 days. The colon cancer was confirmed by the appearance of aberrant crypt foci (ACF) in the colons of the experimental mice. The progression in colon tumor development was correlated with the appearance of the histological biomarker and ACF. Azoxymethane induced colon cancer animals were treated with 400 mg/kg body weight of W. somnifera extract once a week for four weeks orally. After the experimental period, the animals were sacrificed and analyzed for immunocompetent cells, immune complexes and immunoglobulins. W. somnifera significantly altered the level of leucocytes, lymphocytes, neutrophils, immune complexes and immunoglobulins (Ig) A, G and M. The azoxymethane induced colon cancer and immune dysfunction was better controlled by W. somnifera. These results suggested that the immunomodulatory effects of W. somnifera could be useful in the treatment of colon cancer.     

Investigation on the genotoxic effects of long-term administration of sodium arsenite in bone marrow and testicular cells in vivo using the comet assay.

The main source of environmental arsenic exposure in most populations is drinking water in which inorganic forms of arsenic predominate. The single-cell gel electrophoresis technique (the comet assay) measures DNA damage, including double-strand and single-strand breaks, in somatic cells after a variety of genotoxic insults. We have used this method to measure damage to cellular DNA in the bone marrow and testicular cells of mice using the alkaline comet assay for the former and neutral comet assay for the latter. Swiss albino male mice were exposed to sodium arsenite in drinking water at concentrations of 10, 50,100, and 200 mg/l for a period of three months. Concurrently, negative and positive control sets were maintained. The negative control animals were given distilled water as drinking water for the same period of treatment while the animals in positive control sets were either given single or multiple injections of EMS (100 mg/kg body weight) according to the tissue sampled. Following long-term exposure, there was a significant dose-dependent reduction in the size and weight of testes. The comet parameters of DNA, such as tail length (microm), % of DNA in tail, and Olive tail moment (arbitrary units) were increased in both bone marrow and testicular cells due to arsenic-induced DNA strand breaks. A positive dose response relationship was noted. The magnitude of DNA strand break was more pronounced in the bone marrow cells than in the testicular cells. The minimum effective concentrations for inducing DNA damage in bone marrow cells and testicular cells were 10 mg/l and 50 mg/l, respectively. The results of the study indicate that arsenic in drinking water is genotoxic in mice and the comet assay can be used for examining DNA damage in testicular cells as a parameter for evaluating male reproductive toxicity.     

Early and late effects of wound healing on development of colon tumours in a model of colon carcinogenesis by 1,2-dimethylhydrazine in the rat

The early and late effects of wound healing on tumour development at a distant site were evaluated morphologically in an experimental model of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Random bred male Wistar rats were given subcutaneous injections of DMH (20 mg/kg) or saline, once a week, for eight weeks. One week after the last DMH injection the animals received a full thickness skin wound in their dorsal skin and the wound was left open to heal by second intention. Control and DMH treated rats, with or without skin wounds were sacrificed at the twelfth and twentieth weeks. The colons were removed and the incidence, distribution and morphology of any tumours were recorded. Tumours induced by DMH in the colonic mucosa increased in size during the experiment. At the twelfth week, just after healing of the skin wound was complete, the total number of tumours in the colonic mucosa and the number of tumours per rat was significantly higher in the skin-wounding DMH-treated group than in the unwounded group. No differences on tumour incidence and multiplicity were observed among the groups at the twentieth week. Histologically the number of poorly differentiated mucin-secreting carcinomas was increased in the skin-wounding DMH-treated group than in the unwounded group at the twelfth week. This effect was not observed at the twentieth week. The present results suggest that wound healing enhances tumour development at a distant site. This effect coincides with the period of repair and does not have a marked impact on later stages of tumour progression.     

Anticarcinogenic efficacy of phytic acid extracted from rice bran on azoxymethane-induced colon carcinogenesis in rats

This study is carried out to determine the potential of phytic acid extracted from rice bran in the suppression of colon carcinogenesis induced by azoxymethane (AOM) in rats. Seventy-two male Sprague-Dawley rats were divided into 6 groups with 12 rats in each group. The intended rats for cancer treatment received two intraperitoneal injections of AOM in saline (15 mg/kg bodyweight) over a 2-week period. The treatments of phytic acid were given in two concentrations: 0.2% (w/v) and 0.5% (w/v) during the post-initiation phase of carcinogenesis phase via drinking water. The colons of the animals were analyzed for detection and quantification of aberrant crypt foci (ACF) after 8 weeks of treatment. The finding showed treatment with 0.2% (w/v) extract phytic acid (EPA) gave the greatest reduction in the formation of ACF. In addition, phytic acid significantly suppressed the number of ACF in the distal, middle and proximal colon as compared to AOM alone (p<0.05). For the histological classification of ACF, treatment with 0.5% (w/v) commercial phytic acid (CPA) had the highest percentage (71%) of non-dysplastic ACF followed by treatment with 0.2% (w/v) EPA (61%). Administration of phytic acid also reduced the incidence and multiplicity of total tumors even though there were no significant differences between groups. In conclusion, this study found the potential value of phytic acid extracted from rice bran in reducing colon cancer risk in rats.     

Evaluation of the malignant potential of aberrant crypt foci by immunohistochemical staining for beta-catenin in inflammation-induced rat colon carcinogenesis

We previously showed that colitis enhanced the development of cancer and aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in Fischer 344 rats. In this study, we examined the effect of two different anti-inflammatory drugs [non-steroidal anti-inflammatory drugs (NSAIDs: Fenbufen) and a platelet activating factor-receptor antagonist (PAF-RA)] on the inflammation-induced rat colon carcinogenesis. Furthermore, we examined the expression and the localization of beta-catenin protein, and the proliferating cell nuclear antigen (PCNA)-labeling index (LI) in ACF and cancer. PAF-RA significantly decreased the incidence of ACF in the rats (p<0.05), but Fenbufen did not affect the incidence of ACF and cancer. In most of the ACF (91%), beta-catenin was localized at the cell membrane like in normal colon epithelium. In about 9% of the ACF, beta-catenin was overexpressed not only on the cell membrane but also in the cytoplasm. In all of the cancer cells, beta-catenin was overexpressed in the nucleus. When we compared the PCNA-LI in the ACF showing normal beta-catenin expression pattern with that in the ACF showing abnormal beta-catenin expression pattern (overexpression in cytoplasm), there was no significant difference of the PCNA-LI in these two different types of ACF. These findings suggest that immunohistochemical staining of ACF for beta-catenin can evaluate the malignant potential of ACF, and that PAF-RA can be used for preventing the development of ACF in inflammation-induced carcinogenesis.     

Involvement of IGF/IGFBP/Erk axis in the exercise-mediated preventive effects on colorectal cancer in rats

Recent studies have indicated that downregulation of insulin-like growth factor (IGF)-1 and its downstream targets are the main mechanisms underlying the anti-cancer impact of exercise. Therefore, we examined the impact of exercise on chemically induced-aberrant crypt foci (ACF), the earliest step of colorectal carcinogenesis, in rats and involvement of the IGF-1/IGF binding protein (IGFBP)-3/Erk axis. Twenty-four male Wistar rats were assigned into two groups (n=12): the control and exercise group. After eight weeks of training intervention, 6 rats were randomly selected from each group and received four injections of 1,2-dimethylhydrazine (DMH; 40 mg/kg), for two weeks. 0.2% methylene blue staining was used to evaluate the number of ACF in the colon. IGF-1 and IGFBP-3 protein levels in the serum were measured using commercially available ELISA kits for rat. The expression levels of proliferating cell nuclear antigen (PCNA), Erk1/2 and p-Erk1/2 were evaluated in colon tissue. Histological assessments were also performed in all groups. We found that the total number of ACF was significantly lowered after eight-week exercise (P<0.05). Moreover, the exercise program downregulated the IGF-1, PCNA, and p-Erk1/2 expressions and upregulated IGFBP-3 expression. Exercise was also found to increase the goblet cell number and improved colon architecture. Our finding demonstrated reduced ACF number in rat colons following exercise training, and this function may be associated with the inhibition of IGF-1/IGFBP-3/Erk1/2 signaling. Therefore, exercise appears to result in a lower number of ACF for preventing colon cancer.     

Effects of a water-soluble extract of Ganoderma lucidum mycelia on aberrant crypt foci induced by azoxymethane and small-intestinal injury by 5-FU in F344 rats

The present study investigated whether a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (Japanese: Reishi or Mannentake) (designated as MAK) exerted a protective effect against induction of aberrant crypt foci (ACF) by azoxymethane (AOM) and small-intestinal damage induced by the anticancer drug 5-FU. Six-week-old male F344 rats were fed a basic diet (MF), either alone or containing 2.5 % MAK, beginning 1 week before treatment with AOM. The rats were then given subcutaneous injections of AOM (15 mg/kg body weight) once in a week for 3 weeks. Next, beginning 1 day after the final AOM treatment, 25 or 80 mg/kg 5-FU was injected intraperitoneally three times at 5-day intervals. Finally, the rats were killed 3.5 days after the last injection of 5-FU. The large and small intestines were removed, and tissue specimens were examined for both ACF in the large intestine and regeneration of small-intestinal crypts. The number of ACF was significantly decreased by treatment with 25 mg 5-FU and further decreased by 25 mg 5-FU + MAK in comparison with 5-FU alone. Moreover, there was a greater degree of recovery from small-intestinal damage in the 5-FU + MAK groups than in rats that had received 5-FU alone. The present results indicate that MAK ameliorates the colon precancerous lesions induced by AOM and the small-intestinal injury caused by 5-FU, suggesting that MAK could have potential as a preventive agent against colonic precancer, which is a common adverse effect of chemotherapy.     

Modulating effect of Withania somnifera on TCA cycle enzymes and electron transport chain in azoxymethane-induced colon cancer in mice

The aim of the present investigation was to evaluate the efficacy of Withania somnifera on tricarboxylic acid (TCA) cycle enzymes and electron transport chain in azoxymethane-induced experimental colon cancer in mice. Azoxymethane at the dose of 15?mg/kg body weight was induced intraperitoneally once in a week for 28?days. The progression in colon tumor development was correlated with the appearance of the histological biomarker and aberrant crypt foci (ACF). Azoxymethane-induced colon cancer animals were treated with 400?mg/kg body weight of W. somnifera once in a week orally for 28?days. After the experimental period, the animals were killed and analyzed for TCA cycle key enzymes, such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and α-keto glutarate dehydrogenase (α-KGDH). The modulating effect of W. somnifera on TCA cycle key enzymes and electron transport chain complexes were investigated against colon cancer induced by azoxymethane in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as ICDH, SDH, MDH, and α-KGDH in colon cancer bearing animals were observed. W. somnifera administration normalized these enzyme levels in azoxymethane-induced experimental mice. These results suggested that W. somnifera is the promising chemotherapeutic agent for the treatment of colon cancer.     

Characterization of dysplastic aberrant crypt foci in the rat colon induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

The multistage model of colon carcinogenesis is well established in both humans and experimental animals, and aberrant crypt foci (ACF) are generally assumed to be putative preneoplastic lesions of the colon. However, morphological analyses of ACF have suggested that they are highly heterogeneous in nature and their role in tumorigenesis is still controversial. To better understand the biological significance of ACF in carcinogenesis, morphological and genetic analyses were performed using a rat colon cancer model induced by a food-borne colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). ACF of different sizes were collected at weeks 6, 18, 25, and 32 after three cycles of 2-week PhIP feeding (400 ppm in diet) with 4-week intervals on a high-fat diet, and a total of 110 ACF, representing approximately three-quarters of the total ACF, were subjected to histological evaluation. Thirty (27%) were diagnosed as dysplastic ACF, based on cytological and structural abnormalities of crypts. Dysplastic ACF were detected even at week 6 (0.4 per rat), and the numbers increased slightly at later time points, being 0.8, 1.4, and 0.8 per rat at weeks 18, 25, and 32, respectively. The sizes of these dysplastic ACF varied widely from 1 to 16 crypts and 50% (15 of 30) were composed of less than 4 crypts. Immunohistochemical analysis revealed that 83% (25 of 30) of dysplastic ACF demonstrated beta-catenin accumulation; 22 only in the cytoplasm and 3 in both the cytoplasm and nucleus, the latter manifesting a higher grade of dysplasia as compared with the former. Seven dysplastic ACF harbored beta-catenin mutations at codon 32, 34, or 36 in exon 2, and one had an Apc mutation at the boundary of intron 10 and exon 11. Mutations at these sites were also commonly found in colon tumors induced by PhIP. The results of our present study indicate that dysplastic ACF, which accounted for approximately one-fourth of the total ACF, are preneoplastic lesions of colon cancers induced by PhIP in rats.     

In vitro 1?C-labeled amino acid uptake changes and surface abnormalities in the colon after 1,2-dimethylhydrazine-induced experimental carcinogenesis: protection by zinc

The present study explored the regulatory role of zinc on the in vitro uptake of 1?C-glucose and 1?C-labeled amino acids and on colonic surface abnormalities after 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were segregated into four groups: control, DMH-treated, zinc-treated, and DMH + zinc-treated. Colon carcinogenesis was induced through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 16 weeks. Zinc (in the form of zinc sulfate) was given to rats at a dose level of 227 mg/L in their drinking water. DMH treatment caused a significant decrease in the activities of disaccharidases (sucrase, lactase, and maltase), but a significant increase in the activity of alkaline phosphatase. In vitro uptake of 1?C-D-glucose and the amino acids 1?C-glycine, 1?C-alanine, 1?C-lysine, and 1?C-leucine were significantly higher in the colons of DMH-treated rats. Zinc supplementation of DMH-treated rats resulted in regulating the altered intestinal enzyme activities and in vitro uptake of 1?C-amino acids and 1?C-glucose. Scanning electron microscopy revealed drastic alterations in the colon surface morphology after DMH treatment, which were restored after zinc supplementation. Our results confirm a beneficial effect of zinc against DMH-induced alterations in the colons of rats.     

Chronic exposure to endosulfan induces inflammation in murine colon via β-catenin expression and IL-6 production

Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of β-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant β-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose?50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2?mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20?mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30–42 weeks. Immunohistochemical analysis of the colon also showed that expression of β-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of β-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.     

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days −1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBC^CD59-) or reticulocytes (RET^CD59-) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.     

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short‐term exposure. Part 1: Oral route

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM‐202 and 203) and two precipitated (NM‐200 and ‐201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)‐modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose‐dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells. Environ. Mol. Mutagen. 56:218–227, 2015. © 2014 Wiley Periodicals, Inc.     

A histological study of the promotive effect of diethylstilbestrol on diethylnitrosamine initiated carcinogenesis of liver in rat

A single dose (80 mg/kg) of diethylnitrosamine (DEN) was given orally to castrated female Wistar rats. One week after that one half of the animals were treated with diethylstilbestrol (DES) 3 mg/kg/once a week subcutaneously. The other half of the animals received no any hormone or hormone derivatives. The change of the liver cells in animals treated with DEN alone failed to progress beyond the stage of hepatocellular alterations in foci or neoplastic nodules within 8 months, while most of those animals which received DES treatment after DEN initiation developed hepatocellular carcinomas after 6 months. This result denotes that the DES exerts a definite promotive effect on DEN initiated liver cell carcinogenesis.     

Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276–290, 2020. © 2019 Wiley Periodicals, Inc.