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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Arch Toxicol. 2022 Sep 16;96(12):3257–3263. doi: 10.1007/s00204-022-03381-4

N-acetyltransferase 2 acetylator genotype-dependent N-acetylation and toxicity of the arylamine carcinogen β -naphthylamine in cryopreserved human hepatocytes

Mariam R Habil 1, Raúl A Salazar-González 1, Mark A Doll 1, David W Hein 1
PMCID: PMC9641657  NIHMSID: NIHMS1837660  PMID: 36112171

Abstract

We used cryopreserved human hepatocytes that express rapid, intermediate, and slow acetylator N-acetyltransferase 2 (NAT2) genotypes to measure the N-acetylation of β-naphthylamine (BNA) which is one of aromatic amines found in cigarette smoke including E-cigarettes. We investigated the role of NAT2 genetic polymorphism in genotoxicity and oxidative stress induced by BNA. In vitro BNA NAT2 activities in rapid acetylators was 1.6- and 3.5-fold higher than intermediate (p<0.01) and slow acetylators (p< 0.0001). BNA N-acetylation in situ was 3- to 4- fold higher in rapid acetylators than slow acetylators, following incubation with 10 and 100 μM BNA (p< 0.01). DNA damage was 2- to 3-fold higher in the rapid versus slow acetylators (p< 0.0001) and 2.5-fold higher in intermediate versus slow acetylators following BNA treatment at 100 and 1000 μM, ROS/RNS level was the highest in rapid acetylators followed by intermediate and then slow acetylators (p< 0.0001). Our findings show that the N-acetylation of BNA is NAT2 genotype-dependent in cryopreserved human hepatocytes and our data further documents an important role for NAT2 genetic polymorphism in modifying BNA induced genotoxicity and oxidative damage.

Introduction:

For chemical carcinogenesis, the cellular metabolism of aromatic amines is an important step for bioactivation and their carcinogenic potential (Kemp 2015). Many studies have investigated the genotoxic potential of aromatic amines using bacterial systems (Chung et al. 2006; You et al. 1994) and different animal species (Kemp 2015; McQueen et al. 2003; Neis et al. 1985; Sugamori et al. 2006). However, the extrapolation of animal carcinogenicity data to assess human health can lead to an inaccurate risk assessment in humans. There are many interspecies differences that should be considered such as xenobiotic metabolizing enzymes and their genetic polymorphisms (Nauwelaers et al. 2011). This suggests that there is a need for more experiments using human cells (Brambilla and Martelli 1990).

Although smoking is the main source of exposure to chemicals such as aromatic amines in the general population (Boffetta 2008), occupational and environmental exposure to these chemicals also occurs in the rubber and dye industries, hair dyes use and engine emissions (Besaratinia and Tommasi 2013; Nakano et al. 2021; Skare et al. 2009). Exposures to 2-naphthylamine or β-naphthylamine (BNA) occur primarily through inhalation of cigarette smoke, including E-cigarettes. (Fuller et al. 2018). BNA exposures also occur during the heating of food oils or nitrated polycyclic aromatic hydrocarbons metabolized to BNA (Czubacka and Czerczak 2020). BNA is classified as a group 1 human urinary bladder cancer carcinogen by the International Agency for Research on Cancer (Tomioka et al. 2016).

BNA undergoes N- and/or O-acetylation via N-acetyltransferase 2 (NAT2). O-acetylation leads to formation of nitrenium ions that can bind to DNA forming DNA adducts (Adams et al. 1996; Grant 2021). BNA induces DNA damage in the embryo-fetal chicken liver model (Kobets et al. 2019). DNA damage induced by BNA derivatives also has been attributed to generation of reactive oxygen species (ROS) (Ohnishi et al. 2002) suggesting that ROS generation can be a driving factor for BNA toxicity.

NAT2 is a polymorphic phase 2 metabolizing enzyme that plays a key role in metabolism of many aromatic amines (Fretland et al. 2001). Previous studies report the role of CYP450 in metabolism of many aromatic amines (Chun and Kim 2016; Guengerich and Shimada 1998; Hammons et al. 1997). Recently, the role of NAT2 polymorphism in the metabolism of 4-aminobiphenyl was reported in cryopreserved human hepatocytes (Habil et al., 2020). The role of NAT2 and its polymorphisms on BNA metabolism and toxicity was recently reported in Chinese hamster ovary cells (Habil et al., 2022) but the role of NAT2 polymorphisms on BNA metabolism and toxicity in human hepatocytes is yet to be described. Epidemiological studies suggest association between NAT2 and many types of cancer including breast (Baumgartner et al. 2009), liver (Zhang et al. 2005), colon (Wang et al. 2015), and urinary bladder cancer (Mittal et al. 2004; Vineis et al. 2001; Zhu et al. 2015). Previous studies reported associations between BNA exposure due to smoking or occupation and NAT2 slow acetylator alleles with increased bladder cancer risk (Blackadar 2016; Fontana et al. 2009; Grant 2021; Krech et al. 2017).

A wide range of chemicals are metabolized by the liver (Skare et al. 2009) which express many enzymes responsible for carcinogen metabolism (O’Brien et al. 2004). Cryopreserved human hepatocytes retain liver function for at least 1 week and express phase I and phase II enzymes as well as cofactors at physiological concentrations (Guo et al. 2011; Hu et al. 2018).

Therefore, the objective of this study was to investigate the effect of NAT2 genetic polymorphism on N-acetylation of BNA and the associated genotoxicity and oxidative damage generation in cryopreserved human hepatocytes to investigate different pathways of carcinogen metabolism (Bellamri et al. 2017).

Materials and Methods:

Source and processing of cryopreserved human hepatocytes:

Cryopreserved human hepatocytes were received from Bioreclamation IVT, (Baltimore, MD, USA) and stored in liquid nitrogen until use. Upon removal from liquid nitrogen, hepatocytes were thawed according to the manufacturer’s instructions by warming a vial of the hepatocytes at 37°C for 90 seconds and transferred to a 50 mL conical tube containing 45 mL of InVitroGRO HT medium (Bioreclamation IVT). The suspension was centrifuged at 50 x g at room temperature for 5 min. The supernatant was discarded, and cells were washed once in ice-cold PBS before lysing the cells in ice-cold 20 mM NaPO4, 1 mM dithiothreitol, 1 mM EDTA, 0.2% triton-X-100, 1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin A, and 1 μg/mL aprotinin. The lysate was centrifuged at 15,000 x g for 20 min, and the supernatant was aliquoted and stored at −70°C. To mitigate possible instability of human NAT2, supernatant aliquots were thawed only once and used immediately to carry out the enzymatic reactions.

Determination of NAT2 genotype and deduced phenotype:

Genomic DNA was isolated from pelleted cells prepared from human cryopreserved hepatocyte samples as described above using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. NAT2 genotypes and deduced phenotypes were determined as described previously (Doll and Hein 2001). Controls (no DNA) were run to ensure that there was no amplification of contaminating DNA.

Individuals possessing two NAT2 alleles associated with rapid acetylation activity (NAT2*4) were classified as rapid acetylators; individuals possessing one of these alleles and one allele associated with slow acetylation activity (NAT2*5B, NAT2*6A, and NAT2*7B) were classified as intermediate acetylators, and those individuals that possessed two slow acetylation alleles were classified as slow acetylators. Cryopreserved hepatocytes with rapid, intermediate, and slow NAT2 acetylator genotype were selected at random for measurements of N-acetylation as described below.

Measurement of BNA N-acetyltransferase activity in vitro:

Cryopreserved human hepatocytes were selected at random with rapid, intermediate, and slow acetylator genotypes. Rapid NAT2*4/*4 (n=7); intermediate NAT2*4/*5B (n=6), NAT2*4/*6A (n=3); slow NAT2*5B/*6A (n=6), NAT2*5B/*5B (n=2), NAT2*7B/*7B (n=2), and NAT2*6A/*6A (n=1).

N-acetyltransferase assays containing hepatocyte lysate (< 2 mg of protein/ml), BNA (10 – 100 μM) and acetyl coenzyme A (AcCoA) (1000 μM) were incubated at 37°C for 10 min. The amount of the acetylated products was determined following separation and quantitation by high-performance liquid chromatography (HPLC) as previously described (Habil et al., 2022).

Measurement of BNA N-acetyltransferase activity in situ:

Cryopreserved human hepatocytes were selected at random with rapid, intermediate, and slow acetylator genotypes. Rapid acetylator genotypes NAT2*4/*4 (n=6); intermediate acetylator genotypes NAT2*4/*5B (n=5); and slow acetylator genotypes NAT2*5B/*6A (n=5), NAT2*5B/*7B (n=1), NAT2*5B/*5B (n=1), NAT2*7B/*7B (n=1), NAT2*6A/*6A (n=1). Hepatocytes were thawed and transferred to 50 mL conical tubes containing 12 mL of InVitroGRO CP media. One mL of hepatocyte/media mixture was transferred to each well of 12 well Biocoat® collagen coated plates to allow cells to attach for 24 hours at 37°C. Following culture in growth media for 24 hours, the cells were washed 3 times with 500 μL 1X PBS and replaced with media containing BNA with concentrations of 10 or 100 μM. Hepatocytes were incubated for up to 24 hours after which media was removed and protein precipitated by addition of 1/10 volume of 1 M acetic acid. Media was centrifuged at 15,000 x g for 10 min and the supernatant used to separate and quantitate BNA and N-acetylated BNA by HPLC as previously described (Habil et al., 2022).

γH2AX in-cell western assay:

BNA-induced DNA damage in cryopreserved human hepatocytes was assessed by a γH2AX in-cell western staining protocol using slight modifications of a previously described method (Salazar-Gonzalez et al. 2019). Cryopreserved human hepatocytes from rapid NAT2*4/*4 (n=3); intermediate NAT2*4/*5 (n=2), NAT2*4/*6 (n=1); and slow NAT2*5/*5 (n=2) and NAT2*6/*6 (n=1) acetylators were plated in collagen-coated black/clear bottom 96-well plates (Corning, Corning, NY, USA) at a cell density of 5 × 104 using InVitroGRO CP media supplemented with TORPEDO antibiotic mix (BioIVT, Westbury, NY, USA) and allowed to attach overnight. The next morning media was removed, and the attached hepatocytes were washed with PBS and replaced with fresh pre-warmed media containing BNA (0.1 −1000 μM) and incubated for 24 hours. Media were removed, and γH2AX in-cell western staining protocol was performed as follows; hepatocytes were fixed to the plate using 3.7% formaldehyde and incubated at room temperature for 20 min. Then, the cells were permeabilized by washing five times with 0.1% Triton X-100 in TBS. After permeabilization, the cells were blocked using FISH Gelatin Blocking Agent (Biotium, Fremont, CA, USA) diluted in TBS for 90 min at room temperature with constant agitation. Primary antibody anti-phospho-histone H2AX (Ser139) Antibody (Millipore-Sigma, St. Louis, MO, USA) was diluted to 1:1600 and added to the hepatocytes then incubated overnight at 4 °C. The next morning cells were washed with 0.1% Tween 20 in TBS for 5 min, five times. Secondary antibody IRDye® 800CW goat anti-rabbit IgG (LI-COR, Lincoln, NE, USA) was used at a 1:1500 dilution and DNA dye RedDot™ 2 diluted to 0.1X (Biotium, Fremont, CA, USA) to normalize for DNA content. Hepatocytes were incubated with this combination for 60 min and washed again with the Tween 20 solution as previously described. DNA and the γH2AX were simultaneously visualized using an Odyssey CLx imaging system (LI-COR, Lincoln, NE, USA) with the 680 nm fluorophore (red) and the 800 nm fluorophore (green). Relative fluorescent units for γH2AX per cell (as determined by γH2AX divided by DNA content) were divided by untreated cells.

ROS/RNS assay

Cryopreserved human hepatocytes from rapid NAT2*4/*4 (n=3); intermediate NAT2*4/*5 (n=2), NAT2*4/*6 (n=1); and slow NAT2*5/*5 (n=2), NAT2*6/*6 (n=1) acetylators were plated in collagen-coated black/clear bottom 96-well plates (Corning, Corning, NY, USA) at a cell density of 5 × 104 using InVitroGRO CP medium (BioIVT), supplemented with TORPEDO antibiotic mix (BioIVT, Westbury, NY, USA) and allowed to attach overnight. The next morning media was removed and attached cells were washed with PBS and loaded with 20 μM of CM-H2DCFDA (Thermo Fisher, Waltham, MA, USA) for 60 min at 37°C. Afterward, cells were washed 3 times with PBS and culture media treated with increasing concentrations (5-100 μM) of BNA for 4 hours. Treatment with 1 mM H2O2 was used as a positive control. Fluorescence intensity was measured at Ex/Em. = 485/528 nm.

Statistical Analyses:

Differences in N-acetylation rates, genotoxicity, and oxidative damage among hepatocytes expressing different NAT2 genotypes were tested for significance using one-way or two-way analysis of variance (ANOVA) followed by Tukey post-hoc test. All analyses were done using GraphPad Prism 9 (San Diego, CA, USA).

Results:

N-acetylation:

BNA NAT2 activities differed between rapid, intermediate, and slow acetylators (p< 0.0001). BNA NAT2 activity was 1.6- and 3.5-fold higher than intermediate and slow acetylators (p< 0.01 and p< 0.0001 respectively). Intermediate acetylators had higher NAT2 activity than slow acetylators (2.2-fold, p< 0.05) (Figure 1).

Figure 1:

Figure 1:

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In vitro N-acetyltransferase catalytic activities towards BNA. Data illustrates mean ± SEM in rapid (n=7), intermediate (n=9), and slow (n=11) acetylators. Statistical significance was determined using two-way ANOVA followed by a Tukey’s post-hoc test. BNA N-acetyltransferase activities differed significantly between rapid, intermediate, and slow acetylators at 100 μM. *= p< 0.05, **= p< 0.01, ****= p< 0.0001

BNA N-acetylation was concentration-dependent (Figure 2) and differed significantly in situ between rapid and slow; and between intermediate and slow acetylators (p< 0.01). BNA N-acetylation rate was about 3- to 4-fold higher in rapid acetylator than slow acetylator hepatocytes, following treatment with 10 or 100 μM BNA (Figure 3).

Figure 2:

Figure 2:

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Concentration- and time-dependent N-acetylation of BNA in cryopreserved human hepatocytes in situ. Each data point illustrates the mean ± S.E.M. in cryopreserved human hepatocytes from six individual rapid acetylators.

Figure 3.

Figure 3.

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N-acetylation rate of BNA in situ in cryopreserved human hepatocytes from rapid, intermediate, and slow acetylators. Data illustrates mean ± SEM in rapid (n=6), intermediate (n=5), and slow (n=9) acetylators. Statistical significance was determined using two-way ANOVA followed by a Tukey’s post-hoc test. BNA N-acetylation rates differed significantly at 100 μM. *= p< 0.05, ***= p< 0.001

DNA damage:

BNA-induced DNA double-strand breaks were assessed at multiple doses in rapid, intermediate, and slow acetylator hepatocytes, and significant differences were observed between NAT2 genotypes (p< 0.0001) as shown in figure 4. DNA damage was 2 to 3-fold higher in the rapid versus slow acetylator hepatocytes (p< 0.0001) and 2 to 3 -fold higher in intermediate versus slow acetylator hepatocytes (p< 0.0001).

Figure 4.

Figure 4.

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BNA-induced DNA damage in rapid, intermediate, and slow NAT2 acetylator cryopreserved human hepatocytes treated with BNA (0-1000 μM). Statistical significance was determined using two-way ANOVA followed by a Tukey’s post-hoc test. Data illustrates mean ± SEM of four or five experiments. **** p< 0.0001

ROS/RNS:

BNA induced a concentration-dependent increase in ROS/RNS in the three genotypes (linear trend test showed p< 0.0001). ROS/RNS was significantly different between the three genotypes as shown in Figure 5. ROS/RNS levels were the highest in rapid acetylators followed by intermediate and then slow acetylators (p< 0.0001).

Figure 5.

Figure 5.

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BNA-induced oxidative damage in rapid, intermediate, and slow NAT2 acetylator cryopreserved human hepatocytes. ROS and RNS generation were concentration-dependent in the three genotypes (linear trend p< 0.0001). ROS and RNS production differed significantly between the three genotypes Statistical significance was determined using two-way ANOVA followed by a Tukey’s post-hoc test. Each point illustrates the mean ± SEM of 3 individual experiments performed at concentrations up to BNA 100 μM. ****= p< 0.0001.

Discussion:

In the current study, we showed that BNA N-acetylation in cryopreserved human hepatocytes is NAT2-genotype-dependent in vitro and in situ. These findings are consistent with those recently reported for drugs such as sulfamethazine (Doll et al., 2010); hydralazine (Allen et al. 2017), isoniazid (Doll et al. 2017) and solithromycin (Hein and Doll 2017). It also has been shown with the aromatic amine carcinogens 4,4′-methylenedianiline (Salazar-González et al., 2019) and 4-aminobiphenyl (Habil et al. 2020).

Previous studies investigated N-acetylation of BNA using rabbit or rat hepatocytes, and they showed N-acetylation is the major pathway for BNA metabolism and N-oxidation is a minor pathway (McQueen et al. 1983; Orzechowski et al. 1992). Although BNA also undergoes N- and/or O-acetylation catalyzed by N-acetyltransferase 1 (NAT1), previous studies showed that human NAT2 has a three- to four-fold higher affinity than NAT1 for urinary bladder carcinogens such as BNA. (Hein et al., 1993; Hein, 2006).

BNA N-acetylation was both time and concentration-dependent in situ in cryopreserved human hepatocytes. This is consistent with other previously described studies for isoniazid (Doll et al. 2017) and hydralazine (Allen et al. 2017).

Rapid acetylator hepatocytes showed greater concentration-dependent DNA damage induced by BNA treatment than slow acetylators. This is consistent with a recent study investigating BNA-induced DNA damage in in Chinese hamster ovary cells which express rapid and slow acetylator NAT2 haplotypes (Habil et al., 2022). Similarly, 4,4′-methylenedianiline-induced DNA damage is higher in rapid acetylator compared to intermediate or slow acetylator cryopreserved human hepatocytes (Salazar-Gonzalez et al. 2019).

Previously in vitro experiments in prokaryotic (Hakura et al. 2005) and eukaryotic cell lines (Saletta et al. 2007) have been used to investigate aromatic amine-induced genotoxicity. However, due to the lack of drug-metabolizing enzymes in those systems, they require liver-derived enzyme homogenates or S9 mix which contain active phase I enzymes (Kirkland et al. 2007). This mixture lacks active phase II enzymes which play a role in detoxification of aromatic amines; thus, leading to false positive genotoxicity results, unnecessary follow-up animal studies, and overestimation of risk among humans (Corvi and Madia 2017; Groff et al. 2021).

On the other hand, some of the previous studies were done using animal models. Muller et al reported that 2-aminofluorene induced micronuclei in isolated rat hepatocytes (Müller et al. 1993). Also, 4,4′-methylenedianiline induced liver micronuclei in adult rats after repeated dosing (Sanada et al. 2015). More recently, Lin et al., showed the carcinogenic potential of 4-aminobiphenyl in human liver cell lines (Lin et al. 2022). To our knowledge, this is the first study that investigates the genotoxicity of BNA in cryopreserved human hepatocytes.

BNA led to increased ROS/RNS levels in human hepatocytes that was NAT2-genotype dependent. This is consistent with recent studies in Chinese hamster ovary cells which express rapid and slow acetylator NAT2 haplotypes (Habil et al., 2022). It is also consistent with the NAT2 genotype-dependent DNA damage observed both the Chinese hamster ovary cells (Habil et al., 2022) and in cryopreserved human hepatocytes (current study). ROS is a driving factor for DNA damage and carcinogenesis (Bellamri et al. 2022; Ohnishi et al. 2002)

Previous studies have shown that treatment with aromatic amines such as 4-aminobiphenyl generated concentration-dependent increases in ROS in human liver cell models HepG2, Hep3B and L-02 cells (Lin et al. 2022; Lin et al. 2020; Wahyuni et al. 2021). Also, 2-amino-9H-pyrido[2,3-b]indole which is a heterocyclic amine formed during combustion of tobacco and cooking meats until well-done, produced ROS in human hepatocytes (Pathak et al. 2015). More recently, tobacco smoke condensate, which contains BNA, induced ROS in human RT4 bladder cells (Bellamri et al. 2022). However, none of these studies investigated the effect of NAT2 genotype on the induction of ROS or RNS.

In conclusion, our findings revealed an important role of NAT2 genetic polymorphism in N-acetylation of BNA and its genotoxic and oxidative damaging effect in human hepatocytes.

Acknowledgements:

This work was partially supported by United States Public Health Service Grants P20-GM113226, P30-ES030283 and P42-ES023716.

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

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