A comparison of the inflammatory response to a polydimethylsiloxane implant in male and female Balb/c mice.

The implantation of biomaterials elicits a host response that influences the long-term behavior of implanted medical devices. This foreign body response is governed by cells of the immune system. Because sexual dimorphism in the immune system is well-established, a comparative study of the foreign body response in male and female mice was initiated. Eight-week-old male and female Balb/c mice received two subcutaneous implants in the interscapular region of a smooth peroxide-catalyzed polydimethylsiloxane (PDMS) and were sacrificed at 2, 14, 42, 70, and 105 days after implantation (n = 6 per sex per time point). Controls for each time point underwent the surgical procedure but received no implant. Tissue from the implant or surgical site was fixed, processed, and paraffin-embedded for histopathological evaluation and immunohistochemical (IHC) staining for tumor necrosis factor-alpha TNF-alpha) and interleukin-1 beta (IL-1beta). In control animals, an inflammatory response was observed at 2 days that was decreased by 14 days and absent after 42 days. At 2 and 14 days after PDMS implantation, a mild to moderate inflammatory reaction was observed around implants. The peak response was seen at 14 days, and granulation tissue, composed primarily of fibroblasts, macrophages, and neutrophils, was first observed at this time. After 105 days, the implantation site was surrounded by mature connective tissue, which had minimal numbers of macrophages and neutrophils, with severity scores that did not differ significantly in males and females. The immunostaining for TNF-alpha and IL-1beta followed similar temporal patterns, with both reaching a peak at the two week time point and remaining elevated, compared to level of expression in the controls, throughout the 105 day observation period. Staining for both cytokines in the implanted animals was generally higher in females than in males, although this difference was significant only for IL-1beta. These results suggest subtle differences between males and females in the activity of peri-implant inflammatory cells.

Differences in the response to oxidative stress and mutant frequency in CD (Sprague-Dawley) and Fisher 344 rats due to an induced inflammatory response.

In this study, the rodent air pouch model was used to examine the production and processing of oxidative DNA damage in two strains of rats commonly used in toxicity testing. An inflammatory response was induced by injecting zymosan A (50 mg) into an air pouch on male CD (Sprague-Dawley [S-D]) and Fisher 344 (F-344) rats, and the animals were then sacrificed at 1, 3, 7, 14, and 28 days (n = 6 per time point per strain). Tissues from the lining of the air pouch were collected for 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis and for paraffin embedding. Significant (P < 0.01) increases in 8-OH-dG were observed after 1 day in the DNA from cells lining the air pouch of zymosan A-treated versus control S-D (101.5 +/- 27.1 vs. 23.1 +/- 2. 7 8-OH-dG/dG x 10(5)) and F-344 (51.4 +/- 5.3 vs. 14.4 +/- 0.6 8-OH-dG/dG x 10(5)) rats. By 28 days, 8-OH-dG levels had returned to background in S-D rats, but remained elevated in F-344 rats. The frequency of apoptosis was evaluated using the in situ end-labeling (TUNEL) assay, which revealed that zymosan A-treated S-D rats had a significantly (P < 0.05) higher frequency of apoptosis compared to zymosan A-treated F-344 rats. To examine the potential consequences of these differences in endogenously produced DNA damage and apoptosis, we measured mutations at the hprt locus in fibroblasts of the pouch lining and observed a significant (P < 0.05) increase in the mutant frequency at day 28 in F-344 rats (54.2 +/- 13.6 mutants per 10(6) cells) compared to controls (4.5 +/- 2.0 mutants per 10(6) cells). The mutant frequency was not increased in S-D rats. These data demonstrate that strain differences in the production and processing of oxidative DNA damage due to an inflammatory response may impact the long-term pathologic consequences of chronic inflammation. Environ. Mol. Mutagen. 35:336-342, 2000 Published 2000 Wiley-Liss, Inc.

Metabolism and DNA binding of the environmental colon carcinogen 6-nitrochrysene in rats.

The environmental contaminant 6-nitrochrysene (6-NC) has been shown to induce adenomas and adenocarcinomas in the colons of rats. The present study aimed at providing a better understanding of mechanisms that are responsible for this effect. Three female CD rats were injected i.p. with [3,4,9,10-3H]6-NC [9 mumol/rat (346 microCi/rat)], and urine and feces were collected daily for 3 days. In the first 24 h, radioactivity corresponding to 1.3% of the dose was excreted in the urine, whereas 23.0% was recovered in the feces. After 3 days, the total excretions in urine and feces were 2.8% and 34.9% of the dose, respectively. Radioactivity measured in various organs 3 days after injection of [3,4,9,10-3H]6-NC amounted to 24.8% of the administered dose. Fecal metabolites were identified, based on comparison of their chromatographic characteristics with those of standards, as trans-1,2-dihydro-1,2-dihydroxy-6-NC, chrysene-5,6-quinone, and 6-aminochrysene (6-AC); the structure of the latter was further confirmed by mass spectrometry and UV spectral analysis. Metabolites identified in the urine were 6-AC, trans-1,2-dihydro-1,2-dihydroxy-6-NC, and trans-9,10-dihydro-9,10-dihydroxy-6-NC in free forms and also as glucuronide and/or sulfate conjugates. The 32P-postlabeling assay was used to determine the metabolic pathways that were leading to DNA adduct formation in the target (colon) and nontarget (liver, lung, and mammary tissues) organs of female CD rats injected with 6-NC under conditions identical to those of the bioassay (total, 14.8 mumol/rat; single i.p. injections on days 1, 8, 15, 22 and 29). Twenty-four h after the last carcinogen administration, the levels of the adduct derived from trans-1,2-dihydro-1,2-dihydroxy-6-AC were higher than those derived from N-hydroxy-6-AC in all organs examined; however, the highest levels of DNA adducts were found in the lung and not in the target organ, the colon. Although the role of each adduct in colon carcinogenesis needs to be determined, the results favor the ring oxidation and nitroreduction combination pathway as the primary contributor to the activation of 6-NC as a colon carcinogen in the rat.

Assessment of DNA adducts and the frequency of 6-thioguanine resistant T-lymphocytes in F344 rats fed 2,4-toluenediamine or implanted with a toluenediisocyanate-containing polyester polyurethane foam.

Toluenediamines have been of toxicological concern because of their industrial use as intermediates in polyurethane synthesis and because of the potential of their release from degradation of the Microthane polyesterurethane covering of some breast implants. In this study, we have assessed the extent of DNA damage in rats treated with a carcinogenic toluenediamine isomer, 2,4-toluenediamine (2,4-TDA), under conditions that result in tumor induction, and in rats implanted with Microthane polyesterurethane foam. Time and dose-dependent formation of adducts was observed in DNA from the liver and mammary gland of rats fed 10, 40, 80 and 180 ppm 2,4-TDA for up to 6 weeks. In assays conducted 1 to 32 weeks after the start of treatment, no adducts were detected in the DNA of T-lymphocytes isolated from the spleens of animals fed 40 or 180 ppm 2,4-TDA, nor was there an increase in mutations at the hprt locus in these lymphocytes. In rats fed 40 or 180 ppm, 2,4-TDA for 6 weeks, adducts were detectable in DNA isolated from liver and mammary gland for 26 to 43 weeks after termination of the treatment. No DNA damage, as assessed by both DNA adduct measurement and induction of T-lymphocyte hprt mutations, was observed in rats up to 42 weeks after receiving subcutaneous implants of polyesterurethane foam (67 or 267 mg/kg). Although 2,4-TDA is clearly capable of damaging DNA, the results of this study are consistent with the conclusion that Microthane foam-containing implants present a minimal risk of genotoxicity through release and subsequent metabolic activation of 2,4-TDA. The study also indicates that DNA adduct formation and mutation induction in lymphocytes are inadequate biomonitors for measuring exposure to toluenediamines.

Relationships of DNA adduct formation, K-ras activating mutations and tumorigenic activities of 6-nitrochrysene and its metabolites in the lungs of CD-1 mice.

6-Nitrochrysene (6-NC), an environmental pollutant and a potent mouse lung carcinogen, is activated by two major metabolic pathways to yield DNA adducts derived from either trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (1,2-DHD-6-AC) or N-hydroxy-6-aminochrysene (N-OH-6-AC). While the former pathway has been shown to be the major activation pathway leading to DNA adducts in mice treated with 6-NC, the potential contribution of the minor nitroreduction pathway to tumorigenicity in this system is not clear. To evaluate the roles of these activation pathways and the resulting DNA adducts in mouse lung tumorigenesis, we studied DNA adduct formation, the induction of tumors and tumor K-ras mutational spectra in the lungs of male CD-1 mice treated with 6-NC and its metabolites. 6-NC, 6-AC and 1,2-DHD-6-AC produced predominantly a single chromatographically identical dG adduct, and 6-nitrosochrysene (6-NOC) gave a single major adduct that was most likely derived from reaction at the C8 position of deoxyadenosine. 6-NC-, 1,2-DHD-6-AC- and 6-NOC-treated mice developed both adenomas and adenocarcinomas in the lung, whereas only lung adenomas were observed in 6-AC-treated animals. K-ras mutations in adenomas resulting from 6-NC and its metabolites were primarily at G:C basepairs in codons 12 and 13, while adenocarcinomas had K-ras mutations distributed between codons 12, 13 and 61, and involved both G:C and A:T basepairs. The K-ras mutational spectra in codons 12 and 13 were similar in both adenomas and adenocarcinomas whereas a higher percentage of mutations at A:T in codon 61 was found in adenocarcinomas. These results support the conclusion that the 1,2-DHD-6-AC-derived adduct is associated with both adenoma and adenocarcinoma formation and is the primary lesion involved in the induction of mouse lung tumors by 6-NC. The major adduct detected after 6-NOC treatment, which is derived from N-OH-6-AC, is apparently less efficient as an inducer of mouse lung tumors and is associated more specifically with adenocarcinoma formation.

Ethynylestradiol protection against methyl insufficiency in castrated male Wistar/Furth rats fed a methionine-choline-deficient diet.

The interactive effects of dietary methyl insufficiency and the estrogenic compound ethynylestradiol (EE) on the levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were examined in the liver, lungs and pancreas of rats. In addition, such effects on the hepatic content of 5-methyl-deoxycytidine (5-MC) in nuclear DNA were determined. Castrated male Wistar/Furth rats were fed various levels of EE in either: (i) a complete, amino acid-defined diet (diet 1); (ii) the same diet lacking in choline and methionine and supplemented with 0.9% of DL-homocystine (equimolar to methionine) (diet 2); or (iii) diet 2 but only with 0.3% DL-homocystine (diet 2M). Methyl deficiency and EE each independently produced decreased weight gains and increased relative liver weights (liver weight relative to total body weight) compared with control animals. Livers from rats fed diets 2 and 2M without EE had lower levels of SAM and lower SAM:SAH ratios than did the livers from diet 1-fed rats not treated with EE. Hepatic SAM:SAH ratios in diet 1-fed rats were not altered by EE treatment. However, EE treatment increased the hepatic contents of SAM and restored the SAM:SAH levels to normal in rats fed diet 2 or 2M. The levels of SAM + SAH in the livers of rats fed the low homocystine diet (diet 2M) were less than in those fed either diet 1 or diet 2. Thus, the addition of EE at 10 p.p.m. gave protection against reduced levels of SAM, and reduced SAM:SAH ratios in the liver, but had little effect when added to the methyl-adequate diet. No differences in hepatic 5-MC levels were observed in any of the groups as a result of either methyl deficiency or EE treatment. Methyl deprivation alone caused no discernible difference in pancreatic SAM levels but did result in a significant rise in SAH levels and thus in decreased SAM:SAH ratios. EE had no consistent effect on pancreatic SAM, SAH or SAM:SAH ratios in any of the diet groups examined. Similarly, the chronic feeding of diet 2, diet 2M or of EE had no significant effect on the SAM contents of lungs, compared with the corresponding levels in control rats. The protection conferred by EE against SAM insufficiency in the livers of rats fed a methionine- and choline-deficient diet is consistent with the relative insensitivity of female rats to the hepatotoxicity of dietary methyl insufficiency.