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The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: −0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%–2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern. [ABSTRACT FROM AUTHOR]

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration–response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control. [ABSTRACT FROM AUTHOR]

Objective Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. Methods Primary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated β-galactosidase (SA-β-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors. Results We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-β-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found. Conclusions B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process. [ABSTRACT FROM AUTHOR]

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc+/− colon fibroblasts have been previously characterized: however, little is known about their behavior at very early‐stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc+/−) of Pirc rats (F344/NTac‐Apcam1137) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age‐matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P <.001) and delayed replicative senescence onset (P <.05) compared to WCF, along with a lower level of oxidative DNA damage (P <.05). Furthermore, a constitutively higher expression of COX‐2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P <.05), accompanied by higher invasive capability (P <.05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P <.05). However, they neither expressed CAFs markers (α‐SMA, IL‐6) nor responded to CAFs activating stimuli (TGF‐β). Accordingly, CAFs markers and activating stimuli resulted down‐regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc+/− colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients. What's new? Heterozygous mutations in APC represent the earliest event in sporadic colorectal carcinogenesis onset and cause familial adenomatous polyposis syndrome. However, the role of APC‐mutated fibroblasts remains unclear. Here, Apc+/‐ fibroblasts isolated from apparently‐normal colon tissue of Pirc rats showed proliferative, inflammatory features and resistance to oxidative DNA damage, although they did not show cancer‐associated fibroblast features. These data suggest that, at the very early stages of colon tumourigenesis, Apc‐mutated colon fibroblasts favour the establishment of a pro‐tumourigenic environment for pre‐neoplastic lesion development. Early pharmacological targeting of these dysfunctions might be valuable for tumour prevention in familial adenomatous polyposis patients. [ABSTRACT FROM AUTHOR]

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc +/- colon fibroblasts have been previously characterized: however, little is known about their behavior at very early-stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc +/- ) of Pirc rats (F344/NTac-Apc am1137 ) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age-matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX-2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α-SMA, IL-6) nor responded to CAFs activating stimuli (TGF-β). Accordingly, CAFs markers and activating stimuli resulted down-regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc +/- colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)