Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX

Abstract

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3’-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

INTRODUCTION

The potential risk of carcinogenicity to humans has been evaluated for various types of chemicals, such as industrial materials, medical drugs, food additives, and pesticides, using long-term bioassays in rodents. However, standard carcinogenicity tests are not only time-consuming, but also have high costs and require large numbers of animals (Cohen et al., 2019). Multiple new chemical substances are developed every year, but those with low exposure or production are not subject to long-term safety evaluations and are often used without testing for carcinogenicity. Therefore, development of novel bioassays that can efficiently detect carcinogenicity in short-term studies are urgently needed to overcome these problems and contribute to improved animal welfare.

γ-H2AX is the phosphorylated form of the histone constituent protein H2AX that has a phosphorylation at serine 139 and is a well-established biomarker of DNA damage, particularly DNA double-strand breaks (DSBs) (Rogakou et al., 1998). DSBs can be caused by various factors, including reactive oxygen species, replication stress, and defects in DNA repair, as well as direct interactions with ionizing radiation and certain genotoxic agents. In addition, DSBs can be derived from other DNA lesions, such as single-strand breaks (Bonner et al., 2008). ATM (ataxia telangiectasia mutated) is primarily responsible for the phosphorylation of H2AX, but during replication or under hypoxic conditions, ATR (ataxia telangiectasia and Rad3-related), a kinase that recognizes single-strand breaks, can substitute for this role (Falck et al., 2005). γ-H2AX rapidly accumulates not only at the DSB site but also in surrounding chromatin regions (Rogakou et al., 1999), to trigger aggregation of repair proteins (Kinner et al., 2008). This process can be detected microscopically by immunofluorescence and immunohistochemistry using specific primary antibodies (Redon et al., 2012). Thus, immunostaining for γ-H2AX is expected to be a useful tool for predicting genotoxicity and carcinogenicity of chemical substances (Motoyama et al., 2018; Kopp et al., 2019; Plappert-Helbig et al., 2019).

We have recently developed an early detection method for urinary bladder carcinogens that uses γ-H2AX immunostaining and has high sensitivity and specificity (Toyoda and Ogawa, 2022). We demonstrated that 28-day administration of bladder carcinogens to rats significantly increased the number of γ-H2AX-positive bladder urothelial cells, whereas carcinogens that do not target the urinary bladder did not cause such changes. The application of γ-H2AX immunostaining for early detection of carcinogens has also been investigated in other organs, such as lung and stomach (Ying et al., 2018; Okabe et al., 2019). However, this approach has not been fully investigated for the liver and kidney, which are the primary target organs for chemical carcinogenicity. Therefore, in this study we examined γ-H2AX formation in the liver of rats treated with different chemicals for 28 days to investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens.

MATERIALS AND METHODS

Chemicals

We evaluated five hepatocarcinogens: N-nitrosodiethylamine (DEN; CAS no. 55-18-5), di(2-ethylhexyl) phthalate (DEHP; CAS no. 117-81-7), 1,4-dioxane (DO; CAS no. 123-91-9), 3,3’-dimethylbenzidine dihydrochloride (DMB; CAS no. 612-82-8), and thioacetamide (TAA; CAS no. 62-55-5), as well as two non-hepatocarcinogens: 4-chloro-o-phenylenediamine (COP; CAS no. 95-83-0) and N-ethyl-N-nitrosourea (ENU; CAS no. 759-73-9). The suppliers and purities of the chemicals were as follows: DEN (Tokyo Chemical Industry [TCI], Tokyo, Japan; lot no. FBMVM, 99.9%), DEHP (TCI; lot no. Y6X2I, 99.6%), DO (TCI; lot no. MUFZH, 100.0%), DMB (TCI; lot no. WL4IL, 99.1%), TAA (Wako Pure Chemical Industry, Osaka, Japan; lot no. SAJ7861, 99.2%), COP (Sigma-Aldrich, St Louis, MO, USA; lot no. 14606EDV, 98.4%), and ENU (Sigma-Aldrich; lot no. MKBV5750V, 52% [42% water and 3% acetic acid]).

Experimental animals

Five-week-old male F344/DuCrlCrlj rats were obtained from Charles River Laboratories Japan (Yokohama, Japan) and used after 1 week of acclimation. The animals were housed in plastic cages with soft chip bedding in a room with a barrier system controlled for the light/dark cycle (12 hr), ventilation (air exchange rate 18 times/hr), temperature (23°C ± 2°C), and relative humidity (55%  ±  5%). The cages and chip bedding were exchanged twice a week. All animals had free access to a basal diet (CRF-1; Oriental Yeast, Tokyo, Japan) and water with or without the test chemical.

Study design

The present study was performed as two divided experiments using the same protocols (experiments 1 and 2). At the beginning of the experiments, animals were randomly allocated to seven groups of five rats each based on their body weights that were measured immediately before starting the chemical treatment. The animals were administered 0.001% DEN, 1.2% DEHP, 0.5% DO, 1% COP, and 0.001% ENU in experiment 1 and 0.1% DMB and 0.04% TAA in experiment 2 in the basal diet (DEHP, COP, and TAA) or drinking water using light-shielded bottles (DEN, DO, ENU, and DMB) for 28 days. We set the administration doses as the carcinogenic dose in the carcinogenicity test or the maximum tolerated dose for short-term studies, based on published data (IARC, 1978, 1999, 2013, 1974; Weisburger et al., 1980; NTP, 1991; Maekawa et al., 1984). The diet and water were changed once and twice per week, respectively, and body weight and the amounts of supplied and residual diet were measured once weekly during the experimental period. At the end of the experimental period, the animals were exsanguinated under deep anesthesia by inhalation of isoflurane and subjected to laparotomy with excision of the liver. The livers were weighed and a portion was collected and stored at -80°C for extraction of total RNA. The experimental design was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences, Japan, and the animals were cared for in accordance with institutional guidelines.

Immunohistochemistry

For immunohistochemical examination, the livers (left and intermediate lobes) were embedded in paraffin after fixation with 10% neutral-buffered formalin for 72 hr. Serial sections (4 μm thick) were deparaffinized, hydrated, and autoclaved in 10 mM citrate buffer (pH 6.0) for 15 min at 121°C for antigen retrieval. For inactivation of endogenous peroxidase activity, all sections were immersed in 3% H₂O₂/methanol solution for 10 min at room temperature. After blocking nonspecific reactions with 10% normal goat serum, the sections were incubated with primary antibodies targeting γ-H2AX (anti-phospho-histone H2A.X [Ser139] rabbit monoclonal antibody; Cell Signaling Technology, Danvers, MA, USA), Ki67 (anti-Ki67 rabbit monoclonal [SP6] antibody; Abcam, Cambridge, UK), and cleaved caspase-3 (anti-cleaved caspase-3 [Asp175] rabbit polyclonal antibody; Cell Signaling Technology) overnight at 4°C. Visualization of antibody binding was performed using a Histofine Simple Stain Rat MAX PO(R) Kit (Nichirei Corp., Tokyo, Japan) and 3,3’-diaminobenzidine. All sections were counterstained with hematoxylin. γ-H2AX-, Ki67-, and cleaved caspase-3-labeled hepatocytes were counted under a light microscope. All hepatocytes in a randomly-selected field of view in the left lobe were counted and repeated until the total reached 1,000 cells. Then, the number of positive cells per 1,000 cells (γ-H2AX and Ki67) or mm² (cleaved caspase-3) was calculated.

Real-time RT-PCR analysis

Total RNA from the liver was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was generated using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative PCR analysis of mRNA expression was carried out using a 7900HT FAST Real Time PCR System (Applied Biosystems, Foster City, CA, USA) with TaqMan Fast Universal PCR Master Mix and TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA, USA). The primers for the target genes were as follows: Rad51 (Rn01746886_m1), Brip1 (Rn01750632_m1), Xrcc5 (Rn01401464_m1), and Lig4 (Rn01756691_m1). The expression levels of target genes were calculated according to the relative standard curve method and were determined by normalization to Gapdh (Rn01775763_g1) expression. Data are presented as fold-change in values for liver samples in the treatment groups relative to those in the corresponding control groups.

Statistical analysis

Statistical analysis was performed separately for the two experiments. Quantitative values were expressed as means ± standard deviations. Student’s t-test was employed for comparisons between the corresponding control group and each treatment group. Differences with P values less than 0.05 were considered statistically significant.

RESULTS

In-life parameters

No obvious clinical signs were noted throughout the experiment, and all animals survived until the scheduled necropsy. Body weight gain was significantly reduced in rats receiving COP, DMB, and TAA compared to the corresponding controls at week 4 (Table 1). Daily food intake in the TAA group was slightly lower than that of the control group. Absolute and relative liver weights were significantly increased in the DEHP and COP groups. Significant increases in relative liver weight were observed in the DO, DMB, and TAA groups and decreases in absolute liver weight and in relative weight were detected in the TAA and ENU groups, respectively.

Table 1. Body weight, liver weight, and chemical intake data for male F344 rats

Experiment	Group	Body weight (g)						Liver weight						Daily consumption		Chemical intake
		Initial			Week 4			Absolute (g)			Relative (%)			Diet/water	(g/rat/day)	(mg/kg bw/day)
Experiment 1	Control 1	121.8	±	5.8	241.2	±	7.0	9.32	±	0.41	3.86	±	0.09	Diet	13.7	-
														Water	23.8	-
	0.001% DEN	121.6	±	5.8	239.6	±	9.6	9.03	±	0.38	3.77	±	0.10	Water	24.6	1.3
	1.2% DEHP	122.2	±	7.5	231.9	±	6.5	15.0	±	0.51**	6.48	±	0.06**	Diet	14.0	880
	0.5% DO	121.7	±	4.5	235.3	±	8.2	9.39	±	0.25	3.99	±	0.07*	Water	24.9	641
	1% COP	121.8	±	6.4	207.2	±	8.5**	10.9	±	0.46**	5.24	±	0.10**	Diet	11.9	695
	0.001% ENU	122.1	±	6.6	234.7	±	14	8.60	±	0.72	3.66	±	0.13*	Water	25.0	1.3
Experiment 2	Control 2	124.8	±	4.2	234.0	±	11	8.32	±	0.54	3.55	±	0.10	Diet	14.2	-
														Water	19.7	-
	0.1% DMB	123.2	±	4.8	185.3	±	8.5**	8.43	±	0.55	4.54	±	0.13**	Water	14.0	89
	0.04% TAA	124.6	±	3.4	151.7	±	9.8**	7.11	±	0.54**	4.68	±	0.07**	Diet	7.9	23

Values are mean ± standard deviation. DEN, N-nitrosodiethylamine; DEHP, di(2-ethylhexyl) phthalate; DO, 1,4-dioxane; COP, 4-chloro-o-phenylenediamine; ENU, N-ethyl-N-nitrosourea; DMB, 3,3'-dimethylbenzidine dihydrochloride; TAA, thioacetamide. * and ** indicate significantly different from corresponding control at P < 0.05 and 0.01, respectively.

Immunohistochemistry of γ-H2AX in the rat liver

γ-H2AX formation in hepatocytes of rats that received chemical treatments was investigated by immunohistochemistry (Fig. 1). γ-H2AX-positive cells were distributed throughout the left lobe of the liver in the DEN, DO, and TAA groups, whereas γ-H2AX formation in the other treatment groups remained at the same levels as the corresponding control groups. Although most γ-H2AX-positive cells showed characteristic intranuclear dot-like foci (Fig. 1B, inset), ring-like patterns consistent with the nuclear membrane (Fig. 1D, inset) were frequently observed in the DO group. Quantitative analysis revealed that three of five hepatocarcinogens (DEN, DO, and TAA) induced significant increases in the ratio of total γ-H2AX-positive cells compared with the corresponding controls (Figure 1I). In contrast, a significant decrease in γ-H2AX formation was observed in the DEHP group. The number of γ-H2AX-positive cells showing ring-like patterns was significantly increased in the DO group (Fig. 1J).

Fig. 1

Representative immunohistochemical findings for γ-H2AX in the liver of male F344 rats after 28 days of administration of the indicated hepatocarcinogen. Scale bar = 50 µm. A, Untreated control. B, N-Nitrosodiethylamine (DEN). C, Di(2-ethylhexyl)phthalate (DEHP). D, 1,4-Dioxane (DO). E, 4-Chloro-o-phenylenediamine (COP). F, N-Ethyl-N-nitrosourea (ENU). G, 3,3’-Dimethylbenzidine dihydrochloride (DMB). H, Thioacetamide (TAA). I, Quantitative analysis of γ-H2AX formation (total positive cells) in hepatocytes of rats with chemical treatment. J, Quantitative analysis of γ-H2AX formation (ring-like pattern) in hepatocytes of rats with chemical treatment. γ-H2AX staining was evaluated by determining the average number of γ-H2AX-positive hepatocytes (arrowheads) per 1,000 cells. Values are means ± standard deviations. * and **: significantly different from the corresponding controls at P < 0.05 and 0.01, respectively.

Immunohistochemistry of Ki67 and cleaved caspase-3 in the rat liver

To assess the potential relationship between DNA damage and cell proliferation activity, Ki67 expression in hepatocytes was investigated by immunohistochemistry (Fig. 2). Quantitative analysis revealed that the number of Ki67-positive hepatocytes was significantly increased only in the TAA group (Fig. 2I), particularly in the periportal and midzonal area. None of the other groups showed an increase in Ki67 expression, and the DEHP group exhibited a significant decrease in Ki67-positive hepatocytes.

Fig. 2

Representative immunohistochemical findings for Ki67 in the liver of male F344 rats after 28 days of administration of the indicated hepatocarcinogen. Scale bar = 50 µm. A, Untreated control. B, N-Nitrosodiethylamine (DEN). C, Di(2-ethylhexyl)phthalate (DEHP). D, 1,4-Dioxane (DO). E, 4-Chloro-o-phenylenediamine (COP). F, N-Ethyl-N-nitrosourea (ENU). G, 3,3’-Dimethylbenzidine dihydrochloride (DMB). H, Thioacetamide (TAA). I, Quantitative analysis of Ki67 expression in hepatocytes of rats with chemical treatment. Ki67 staining was evaluated by determining the average number of Ki67-positive hepatocytes per 1000 cells. Values are means ± standard deviations. **: significantly different from the corresponding controls at P < 0.01.

γ-H2AX formation is induced in apoptotic cells as well as in cells with DNA damage. In addition, previous in vitro studies have suggested that the ring-like patterns of γ-H2AX formation are associated with apoptosis, although the relevance in vivo has not been clarified (Bonner et al., 2008; Solier and Pommier, 2014). Thus, to investigate the possible relationship between γ-H2AX formation and apoptosis, immunohistochemical analysis of cleaved caspase-3, a well-known biomarker of apoptotic cells, was performed in the liver of rats treated with DEN and DO that showed significant increases in γ-H2AX formation (Fig. 3). Quantitative analysis revealed that there were no significant increases in the number of cleaved caspase-3-positive cells in the DEN or DO groups (Fig. 3D).

Fig. 3

Representative immunohistochemical findings for cleaved caspase-3 in the liver of male F344 rats after 28 days of administration of the indicated hepatocarcinogen. A, Untreated control. B, N-Nitrosodiethylamine (DEN). C, 1,4-Dioxane (DO). Scale bar = 50 µm. D, Quantitative analysis of cleaved caspase-3 expression in hepatocytes of rats with chemical treatment. Cleaved caspase-3 staining was evaluated by determining the average number of cleaved caspase-3-positive hepatocytes (arrowheads) per mm². Values are means ± standard deviations.

Real-time RT-PCR of DNA repair-related genes in the rat liver

DSB, one of the most serious forms of DNA damage, is mainly repaired by two mutually exclusive pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). As mentioned above, although γ-H2AX is formed through various types of DNA damage, we focused on DSBs as the main cause of γ-H2AX formation in this study. We examined mRNA expression of Rad51/Brip1 and Xrcc5/Lig4 in the rat liver as genes specific for HR and NHEJ, respectively, by real-time RT-PCR analysis (Fig. 4). Significant increases in expression of all four genes were observed in the TAA group, and increased expression of Rad51 was detected in the DEN and DO groups. In contrast, significant decreases in expression of Rad51, Brip1, and Lig4 were observed in the DEHP group.

Fig. 4

Relative mRNA expression of DNA repair-related genes in the liver of male F344 rats treated with hepatocarcinogens for 28 days. DEN, N-nitrosodiethylamine; DEHP, di(2-ethylhexyl)phthalate; DO, 1,4-dioxane; DMB, 3,3’-dimethylbenzidine dihydrochloride; TAA, thioacetamide. Values are means ± standard deviations. * and **: significantly different from the corresponding controls at P < 0.05 and < 0.01, respectively.

DISCUSSION

To evaluate the potential application of γ-H2AX as a biomarker of carcinogenicity in the liver, in this study we examined γ-H2AX formation by immunohistochemistry of rat liver samples derived from animals in 28-day repeated-dose studies. Three of the five hepatocarcinogens tested (DEN, DO, and TAA) significantly increased γ-H2AX formation in hepatocytes, whereas the other two (DEHP and DMB) did not induce this change and had the same levels of γ-H2AX as seen in livers from rats treated with the two non-hepatocarcinogens (COP and ENU).

We previously reported a significant increase in γ-H2AX formation in liver of rats treated with DEN, a genotoxic hepatocarcinogen, for 28 days (Toyoda et al., 2015); this result was confirmed in the present study. Although the number of γ-H2AX-positive hepatocytes was increased by DEN treatment, no increase was observed in the urinary bladder, which is not a carcinogenic target (Toyoda et al., 2015). These results indicate that γ-H2AX immunostaining of rat tissue samples is useful to identify carcinogenic target organs of chemicals. Meanwhile, no significant increase in γ-H2AX formation was observed in DMB-treated rats. It has been reported that DEN forms DNA adducts via metabolism by CYP2E1 in the liver (Verna et al., 1996), and that the gene expression pattern in the liver of DMB-treated rats is similar to that of DEN (Furihata et al., 2018). DMB has been shown to induce benign and malignant tumors in rat liver as well as codon-specific mutations primarily in the H-ras oncogene that are frequently detected in DMB-induced tumors, suggesting that the increased tumor incidence is associated with the genotoxic mechanisms (NTP, 1991). Although there are few certain reports of adduct formation by DMB, Makena et al. suggested that the involvement of oxidative stress in the DMB-induced carcinogenesis appears to be unlikely (Makena and Chung, 2007). Some of the urinary bladder carcinogens that showed false-negative results in the evaluation of γ-H2AX alone could be detected in a combined evaluation with immunostaining for bladder stem cell markers, including aldehyde dehydrogenase 1A1 (Yamada et al., 2021). Thus, in addition to γ-H2AX, a combined evaluation of markers corresponding to liver-specific stem cell characteristics and carcinogenic mechanisms may be useful to increase sensitivity.

Formation of γ-H2AX was also increased by TAA, a nongenotoxic hepatocarcinogen. After metabolized by CYP2E1, TAA is cytotoxic to hepatocytes via oxidative stress and increases reactive proliferative activity (Henderson et al., 2015), as shown by increased Ki67 expression observed in this study. Despite being classified as a nongenotoxic chemical, here we observed that TAA induced significant increases in mRNA expression of DSB repair-related genes, including Rad51, Brip1, Xrcc5, and Lig4, although the effect of mRNA expression on DNA repair process needs to be considered. It has been reported that γ-H2AX formation is induced not only by direct DNA damage, but also indirectly by cell proliferation (Ichijima et al., 2005; Tu et al., 2013). In addition, a previous study analyzing microarray data showed that repeated-dose administration of DEN and TAA induced similar expression patterns of DNA damage-related genes (Omura et al., 2014). Thus, TAA-induced γ-H2AX formation observed in the present study may be associated with secondary DNA damage due to replication stress caused by sustained proliferative stimulation. These results indicate that γ-H2AX immunostaining can detect carcinogenicity of chemicals in the liver regardless of the presence or absence of direct genotoxicity.

Interestingly, DO induced γ-H2AX formation in a ring-like pattern, which clearly differed from typical γ-H2AX foci. Various bladder carcinogens have also been found to induce a similar ring-like pattern of γ-H2AX formation in the bladder mucosa (Toyoda and Ogawa, 2022). Although the ring-like distribution of γ-H2AX consistent with nuclear membranes has been suggested to be associated with apoptosis in vitro (Solier and Pommier, 2014), here we observed no increase in the number of apoptotic hepatocytes in the DO group as indicated by immunostaining for cleaved caspase-3. Oral treatment of DO causes features associated with cytotoxicity in rat liver, such as centrilobular necrosis (Kano et al., 2008; Kano et al., 2009), and liver tumors induced by DO are considered to be related to compensatory regeneration due to its cytotoxic activity (Stott et al., 1981). Although several reports concluded that DO is nongenotoxic based mainly on results of in vitro assays (Morita and Hayashi, 1998; EU, 2002), there is still controversy regarding DO genotoxicity since positive results for in vivo genotoxicity tests have been reported (Gi et al., 2018; Itoh and Hattori, 2019), such as inducing DNA single strand breaks in the rat liver, without the formation of DNA adducts (IARC, 1999). In fact, RNA-Seq and principal component analysis of 11 marker genes, including genes related to DNA damage and apoptosis, revealed that DO induces a characteristic gene expression profile that is distinct from both genotoxic (DEN) and non-genotoxic (DEHP) hepatocarcinogens (Furihata et al., 2018). Thus, further analysis is needed to determine which type of DNA damage is associated with the ring-shaped patterns for γ-H2AX-positive hepatocytes seen in the DO group.

DEHP, a widely used plasticizer in polymer products, is a potent activator of peroxisome proliferator-activated receptor α (PPARα) expression (IARC, 2013). Interestingly, both Ki67-positive cells and mRNA expression of DNA repair-related genes as well as γ-H2AX formation were significantly decreased in the liver of DEHP-treated rats. It has been reported that the increase in the proliferative activity of hepatocytes induced by DEHP administration is transient, and that hepatic enlargement is maintained by sustained PPARα activation (Smith-Oliver and Butterworth, 1987; IARC, 2013). Thus, a shorter-term study may detect an increase in indirect γ-H2AX formation. DEHP is one of the exceptional chemicals that shows false-negative results in the 8-week medium-term bioassay for detection of hepatocarcinogens (Ito et al., 2003). In addition to PPARα, multiple molecular signals, including oxidative stress mediated by tissue macrophages, have been implicated in the carcinogenic mechanism of DEHP (Rusyn and Corton, 2012; IARC, 2013).

In conclusion, three of the five hepatocarcinogens evaluated in this study significantly increased γ-H2AX formation in rat hepatocytes after 28 days of oral administration, whereas the two non-hepatocarcinogens did not induce this change. These results suggest that γ-H2AX immunostaining has the potential to be useful for early detection of hepatocarcinogens, but that it should be used in combination with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

ACKNOWLEDGEMENTS

This work was supported by a grant for Research on Food Sanitation from the Ministry of Health, Labour and Welfare, Japan. The authors thank Ayako Saikawa and Yoshimi Komatsu for expert technical assistance in processing high-quality histological materials.

Conflict of interest

Mizuki Sone was an employee of Kao Corporation (Tochigi, Japan) during the conduct of this study. The authors declare that there is no conflict of interest.

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