NAT2 genetic polymorphism and age dependent ecacy of neurotropin® for the enhancement of aggrecan gene expression in nucleus pulposus cells

Intervertebral disc degeneration, one of the major causes of low-back pain, results from altered biosynthesis/turnover of extracellular matrix in the disc. Previously, we reported that the analgesic drug Neurotropin® (NTP) had an anabolic effect on glycosaminoglycan synthesis in cultured nucleus pulposus (NP) cells via the stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1. However, its effect on the aggrecan core protein was not signicantly detected, because of the data variance. A microarray analysis suggested that the effect of NTP on aggrecan was correlated with N-acetyltransferase 2 (NAT2), a drug-metabolizing enzyme. Specic NAT2 alleles are known to correlate with rapid, intermediate, and slow acetylation activities and side effects of various drugs. We investigated the association between the ecacy of NTP on aggrecan expression and the NAT2 genotype in cell donors.


Page 3/16
Background Intervertebral disc (IVD) degeneration is a chronic and progressive disease. Research on effective medical treatments for this disorder is ongoing. The IVD is a composite of substructures that consists of con ning end plates on the superior and inferior faces, the highly brous anulus brosus on the outer periphery, and the highly hydrated nucleus pulposus (NP) at the center [1]. The IVD appears to be designed to sustain compression loads that are bene cial to it, as loading is the physiological stimulus for matrix turnover and induces matrix synthesis [2][3][4]. However, excessive loading can lead to deleterious changes in the IVD by downregulating the genes encoding anabolic proteins, with signi cant effects on aggrecan formation, while upregulating genes encoding matrix metalloproteinase [4], thereby inducing matrix degradation [5]. Aggrecan is the major noncollagenous component of the IVD. It is a large proteoglycan possessing numerous glycosaminoglycan (GAG) chains and a core protein, and is an integral part of the extracellular matrix in cartilaginous tissues. Its abundance and unique molecular features, i.e., the formation of aggregates in association with hyaluronan, provide the disc with its osmotic properties and ability to withstand compressive loads. The degradation and loss of aggrecan result in impairment of disc function and the onset of disc degeneration [6].
Previously, we reported that the analgesic drug Neurotropin ® (NTP) had an anabolic effect on GAG synthesis in NP cells from the IVD via the stimulation of chondroitin sulfate Nacetylgalactosaminyltransferase 1 (CSGALNACT1), which initiates the synthesis of chondroitin sulfate (CS) polysaccharide chains attached to the core protein of aggrecan [7]. However, in our previous study, NTP did not signi cantly promote the expression of the gene encoding the aggrecan core protein, as it was decreased by the addition of NTP to the cells of a couple of donors. If we anticipate a new application of NTP as a medicine for the restoration of deteriorated disc matrix, NTP should also increase or at least maintain the expression level of the aggrecan core protein, to anchor the increased CS side chains onto the cell surface in association with hyaluronan. To explain the large variance in our previous data, in the current study, we investigated whether the difference in cellular responsiveness to NTP stems from the genetic background of the donors.
NTP, a nonprotein extract of in amed rabbit skin inoculated with the vaccinia virus, has been used in Japan to treat chronic pain via oral, intramuscular, or intravenous administration [8], and was reported to provide effective relief for various types of pain, such as headache, low-back pain, neck-shoulder-arm syndrome, postherpetic neuralgia, and bromyalgia [8][9][10][11]. Despite its clinical advantages, the characteristics of NTP remain unclear regarding two issues: rst, its main active ingredient is unclear because NTP comprises many components, including nucleic acids, amino acids, and sugars [12]; second, the mechanism underlying the local action of this reagent is not clearly understood, although the main effect of NTP has been reported to be the activation of the descending monoaminergic pain inhibitory systems of the central pain pathway [13].
To identify the genetic basis of the large variance in our previous study we re-explored the microarray data generated previously to investigate comprehensively the gene expression changes in NTP-treated NP cells from four patients (all data are available on the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/gds/?term=GSE114169). The gene encoding arylamine Nacetyltransferase 2 (NAT2) appeared to be correlated with cell donor responsiveness to NTP regarding aggrecan gene expression (Fig. 1). NAT2, a drug-metabolizing enzyme, is one of two structurally related isoenzymes, NAT1 and NAT2. These NATs are phase II xenobiotic metabolism enzymes that catalyze the detoxi cation of arylamines via N-acetylation and the bioactivation of N-arylhydroxylamines by Oacetylation. NAT2 acetylates a large variety of arylamine-acceptor structures, such as caffeine, procainamide, and sulfasalazine, as well as the antituberculosis drug isoniazid [14][15][16]. Speci c types of NAT2 alleles are known to be correlated with distinct metabolic activities; patients with a NAT2 that is inactive against isoniazid have been reported to have a higher risk of developing antituberculosis-druginduced liver injury [16][17][18][19][20]. Genotypic polymorphisms at the NAT2 locus give rise to either the "slow" or the "rapid" acetylator phenotype, as well as the "intermediate" acetylator phenotype in "slow/rapid" heterozygotes [21]. These phenotypes also affect individual variation in cancer susceptibility, responses to environmental toxins, and the effectiveness of prescribed medications [22,23].
We therefore hypothesized that genetic polymorphism in NAT2 affects the modulation of the expression of the aggrecan mRNA by NTP treatment, because NTP possibly includes components with arylamineacceptor structures. Our objective was to investigate the association between the promoting effect of NTP on aggrecan gene expression in NP cells and the NAT2 genotype status in the cell donors.

Study population
We conducted a cross-sectional study in the cultured cells donated from 31 Japanese patients (aged 28-68 years) who underwent discectomy at Tokai University Hospital from January 2019 to March 2020. The surgical-waste samples consisted of lumbar IVDs from herniation and vertebral and spinal fusions with two bursts from thoracic and lumbar IVDs. The frequency of females was 38.7%, and the mean age ± standard deviation (SD) of the female and male donors was 52.0 ± 12.2 and 52.5 ± 11.9 years, respectively.

Tissue culture and cell isolation and expansion
The NP tissue was carefully separated from the anulus brosus. We used NP tissue for our experiments when at least 0.05 g of wet weight was obtained. The sample was cut into small pieces and incubated in a-minimal essential medium (a-MEM; FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO), 100 U/ml penicillin (Thermo Fisher Scienti c, Waltham, MA), and 100 mg/ml streptomycin (Thermo Fisher Scienti c) at 37 °C and 5% CO 2 for 2 weeks.
The cultured NP tissues were collected and digested with TrypLE Express (Thermo Fisher Scienti c) for 30 min, followed by incubation with 0.25 mg/ml Collagenase-P (Roche, Basel, Switzerland) for 80 min at 37 °C. The isolated cells were washed twice with a-MEM and seeded at a density of approximately 5 ´ 10 3 cells/cm 2 . Cells were cultured in a-MEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO 2 under hypoxic conditions of 2% O 2 . The medium was replaced twice a week and the cells were trypsinized (Thermo Fisher Scienti c) and subcultured before they reached con uence. Cells harvested from second-passage cultures were used for experiments with NTP; thereafter, further-passaged cells were used to extract genomic DNA.

Chemicals
NTP was provided by Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, Japan). The biological activity of NTP was expressed in NTP Units (NU). l-ascorbic acid 2-phosphate (AsAP) was purchased from FUJIFILM Wako Pure Chemical.

Treatment of cultured NP cells with NTP
NP cells were seeded in 6-well culture plates at a density of 5 ´ 10 3 cells/cm 2 on "day zero" and were cultured in a-MEM containing 10% FBS (basal medium) overnight prior to NTP addition. As reported previously, the cells were stimulated with NTP dissolved in fresh a-MEM supplemented with 10% FBS and 50 mg/ml AsAP. The medium was replaced every second day for 1 week. The NTP concentrations were set at 0.1 and 0.2 mNU/ml. The concentration of NTP in the culture media was set to its approximate levels in the blood plasma when taken according to the clinical prescriptions. On days 6 and 8, NP cells were harvested and the expression of the aggrecan gene was evaluated.

Real-time quantitative PCR
Cells cultured in the presence or absence of NTP treatment were harvested and homogenized in lysis buffer, and total RNA was prepared using an SV Total RNA Isolation System (Promega, Madison, WI). For each sample, 2 mg of total RNA was reverse transcribed into cDNA using a High Capacity RNA-to-cDNA kit (Life Technologies, Waltham, MA). Relative quanti cation of the target mRNA was performed using the comparative C T method with the sets of primers and probes for the endogenous control glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH, Hs99999905_m1), and the target genes of aggrecan (ACAN) (Hs00153936_m1) and CSGALNACT1 (Hs00218054_m1), all of which were provided as predeveloped TaqMan Gene Expression Assay Reagents (Life Technologies). The assay for ACAN covers the mRNAs of transcript variants for the aggrecan core protein, including variants of 1, 2, X2, and X3. The PCR ampli cation and analysis were performed on a QuantStudio 3 real-time PCR instrument (Life Technologies).
NTP was considered effective when the expression of the ACAN mRNA was increased by more than 1.1fold compared with the control culture in the basal medium. For each donor, the highest values obtained in four experimental conditions (treated with two dose settings and harvested at two time points) were selected and analyzed.
NAT2 genotyping and assignment of acetylator phenotypes Subcultured NP cells were lysed and genomic DNA was puri ed using a DNA puri cation kit (NucleoSpin ® Tissue; Macherey-Nagel GmbH, Düren, Germany). DNA was quanti ed using a NANODROP LITE spectrophotometer (Thermo Fisher Scienti c) and was stored at -30 °C until use. Five single-nucleotide polymorphisms (SNPs) were analyzed: rs1041983 (282C > T), rs1801280 (341T > C), rs1799929 (481C > T), rs1799930 (590 G > A), and rs1799931(857 G > A). The genotyping assays using PCR were performed based on a fundamental procedure [24] and the manuals from the reagent provider (Life Technologies). Brie y, a 20´ primer and probe mix from TaqMan Drug Metabolism Assays (Life Technologies) were supplied for each assay, and a 1´ concentration of this mix, as well as a half volume of 2´ TaqPath ProAmp Master Mix (Life Technologies) and 10 ng of the genomic DNA sample, were added to each well. The thermal conditions of the experiment were 95 °C for 5 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The PCR ampli cation and endpoint reading were performed on a QuantStudio 3 real-time PCR instrument. In each experiment, control samples with no DNA template were run to ensure that there was no ampli cation of contaminating DNA. Cell donors possessing rapid/rapid homozygous NAT2 alleles were classi ed as rapid acetylators, individuals possessing a rapid/slow heterozygous genotype were classi ed as intermediate acetylators, and individuals possessing a slow/slow homozygous genotype were classi ed as slow acetylators. The inference of haplotype-phenotype information pertaining to the NAT2 acetylator status was determined based on the annotation table provided in Human NAT2 Alleles (Haplotypes), a database built by Democritus University of Thrace (http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.htm).

Statistical analysis
The calculations of odds ratios (ORs) and Pearson's chi-squared test (to obtain P-values) were performed in 2 ´ 2 tables using the Statistical Analysis WEB-BellCurve software (https://bellcurve.jp/statistics). The calculation of 95% con dence intervals (95% CIs) was carried out using the Microsoft Excel 2016 software according to the equation: where a is the number of responders with variant NAT2 haplotypes, b is the number of nonresponders with variant NAT2 haplotypes, c is the number of responders with the wild-type homozygous NAT2 genotype, d is the number of nonresponders with the wild-type homozygous NAT2 genotype, and "ln" stands for natural logarithm. In addition, the following analyses were performed using the functions of the Microsoft Excel 2016 software: Student's t-test and F-test were applied to compare the differences between two groups regarding phenotype and changes in the levels of the aggrecan mRNA; the correlation coe cient between changes in the aggrecan mRNA after NTP treatment and donor's age was calculated; and Student's t-distribution test for its signi cance was performed. Signi cance was set at P < 0.05.

Compliance with ethical standards
This study, including the use of patient-derived surgical-waste material and sequence analysis of genomic DNA, was approved by the Clinical Research Ethics Committee of Tokai University School of Medicine (study code: 18I-25), and was conducted in accordance with approved protocols. Informed consent forms with written provision were completed by all patients before the donation of IVD samples.

Results
We identi ed four haplotypes, NAT2*4, NAT2*5B, NAT2*6A, and NAT2*7B, in the 31 donors (Table 1). The wild-type NAT2*4 haplotype was present in 79.0% of the examined subjects, whereas the remaining three haplotypes were variants. There was no signi cant difference (Pearson χ2 test) in the distributions of haplotypes between our data and the data from a study that assessed 200 healthy Japanese volunteers [25]. As exceptions, the NAT2*11 and NAT2*13 haplotypes, which are present in 0.25% and 1.25% of the Japanese population, respectively, were not detected in the cell donors enrolled in our study.  Phenotype and age-related changes in the expression of the aggrecan mRNA after NTP treatment The quantitative data pertaining to the changes in the expression levels of the aggrecan mRNA after NTP treatment compared with each control were plotted against the NAT2 phenotypes (Fig. 2a). As shown in the box-whisker plots, there was a large variance in the rapid acetylator phenotype; consequently, no signi cant effect of the NTP treatment was detected compared with the control (mean ± SD, 1.12 ± 0.56, P = 0.187, Student's t-test), while there was a signi cant increase in the intermediate acetylator phenotype (mean ± SD, 1.15 ± 0.16, P = 0.002, Student's t-test). Although the mean values of each phenotype were similar (1.12 and 1.15, respectively), there was a statistically signi cant difference in the distributions of data between the rapid and immediate acetylator phenotypes (P = 2.69E-05, F-test). The change in the expression of the aggrecan mRNA after NTP administration is plotted against donor's age in Fig. 2b. There was a weakly negative correlation with age in all data (r = -0.532, P = 0.0021, Student's tdistribution test). Regarding the rapid acetylator phenotype, the same tendency was found at higher signi cance (r = -0.683, P = 0.002, Student's t-distribution test). In contrast, no signi cant correlation was found for the immediate acetylator phenotype.
Male donors classi ed as having an intermediate acetylator phenotype exhibited a favorable response to NTP treatment. Gender-speci c analyses are shown in Fig. 3a, b. An age-related correlation was not observed in the male donors, while a signi cantly negative correlation was observed in the female donors (r = -0.773, N = 12, P = 0.006, Student's t-distribution test). In Fig. 3c Recon rmation of e cacy of NTP on expression of the CSGALNACT1 mRNA To con rm the e cacy of NTP that we reported previously [7], the changes in the relative expression of the CSGALNACT1 mRNA were examined (Fig. 3d). Ten samples were impartially selected according to the results of NAT2 phenotype (rapid:immediate = 5:5), responsiveness (responder:nonresponder = 5:5), and gender (female:male = 4:6) presented above. Quantitative PCR showed that NTP treatment signi cantly increased the expression of the CSGALNACT1 mRNA in NP cells compared with the control (mean ± SD, 1.28 ± 0.37, N = 10, P = 0.013, Student's t-test).

Discussion
In the current study, we showed that the NAT2 immediate acetylator phenotype (comprising the NAT2*4/*5B, NAT2*4/*6A, and NAT2*4/*7B genotypes) was associated with the effectiveness of NTP regarding the promotion of the expression of the aggrecan mRNA in cultured NP cells. Thus, NAT2 may be one of the genetic factors that act as a watershed that separates the presence or absence of negative effects of NTP in cultured NP cells. In contrast, we did not nd any signi cant differences between immediate and rapid (homozygous for the NAT2*4 allele) acetylator phenotypes regarding their mean values of upregulation of aggrecan mRNA expression (Fig. 2a). This was because a few strongly positive responses by the cells from young donors (<45 years) counterbalanced the negative responses by the cells from older donors (>45 years) in the rapid phenotype group (Fig. 2b). This age-related variance in cellular responsiveness was also found among the female donors (Fig. 3b).
A study of middle-aged and elderly postmenopausal women with exogenous estrogen therapy reported that neither estrogen concentration nor age was correlated with NAT2 activities, as measured by the caffeine metabolic ratio [26]. In another study that enrolled children of various ages, including infants, discordance between phenotype (acetylation) and genotype (NAT2) was reported [27]. In contrast, during the development of the outbred CD-1 mouse strain, a gender-dependent difference was observed; the kidney p-aminobenzoic acid/Nat2-acetylating activity of female mice showed a 2.5-fold increase at day 80 compared with day 1, whereas males showed a 4.3-fold increase at day 25 and a 5.8-fold increase at day 80 [28]. These ndings provided knowledge about the difference between genders and the agerelated changes in the function of NAT2, which currently exhibit diverse aspects; thus, it remains unclear whether any changes occur in age-or gender-speci c manners.
Generally, NAT2 genetic variants have been linked to decreased enzymatic activity and variable stability, leading to an imbalance in the xenobiotic detoxi cation and increased susceptibility to different forms of cancer [22,29]. Nevertheless, the rapid NAT2 phenotype has been reported to metabolically activate the toxicity of xenobiotic substances, such as N-hydroxylated heterocyclic aromatic amines (HAAs) via Oacetylation, to form the reactive N-acetoxy species. Some HAAs are formed when meat is cooked at high temperature for a long time, and high HAA intake has been associated with an increased risk of colorectal cancer compared with the immediate/slow acetylator phenotypes [30]. Therefore, NAT2 with a rapid phenotype seems to activate environmental toxins in some cases, in addition to catalyzing several pharmacologically and toxicologically signi cant detoxi cation reactions [31]. Moreover, a signi cant association between the NAT*6A polymorphism and age-related hearing loss has been reported: the genetic effect on presbycusis stemmed from the observation that NATs, together with cytochrome P450 and glutathione S-transferases, metabolize a wide range of xenobiotics and are important for the balance of oxidative status to protect cells against environmental toxins and the cellular damage caused by oxidative free radicals [32]. Therefore, the arylamine-catalyzing ability of NAT2 combined with other factors of the cultured NP cells may also be implicated in the current observations.
Regarding the functional mechanism underlying the effect of NTP on cultured NP cells, we previously reported that NTP activates the PI3-AKT pathway and promotes the synthesis of sulfated GAGs, such as chondroitin sulfate, heparin sulfate, and keratin sulfate. As one of the key effectors of the function of NTP, we demonstrated an increase in the levels of the CSGALNACT1 enzyme, which initiates the synthesis of CS polysaccharide chains [7]. In the current study, we detected a NAT2 phenotype-dependent increase in the expression levels of the aggrecan mRNA induced by NTP in cultured NP cells. According to a previous report [6], aggrecan abundance reaches a plateau in the early twenties, declining thereafter because of proteolysis, mainly by matrix metalloproteinases and aggrecanases, although the degradation of hyaluronan and nonenzymatic glycation may also participate in this process. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process .
In the current study, the male donors classi ed as having an immediate acetylator phenotype exhibited the highest signi cance in the correlation with the frequency of responders to NTP (OR = 14.0; 95% CI, 1.54-127.2; P = 0.012, Pearson χ2 test). It is noteworthy that no cells from individuals possessing an immediate acetylator phenotype, including donors in their late sixties, were affected negatively by NTP (Fig. 3a, c). Regarding the age-related deterioration of human NP tissue, we previously reported an exhaustion of NP progenitor cells with evidence of an exponential decline in the frequency of Tie2positive cells in freshly isolated cells from NP tissues donated by 23 patients (aged 19-70 years) [33]. Therefore, encouraging NP cells to produce aggrecan via NTP treatment will be bene cial for elderly male patients, even if the effect is NAT2 phenotype-speci c. As we have demonstrated that NTP treatment signi cantly increased the expression of the CSGALNACT1 mRNA in the cells from 10 impartially selected individuals, it is likely that the promoting effect of NTP regarding the expression of CS side chains is also reliable.
Our study had several limitations. First, our sample size was small, especially the number of female individuals who possessed NAT2 variants (N = 3), which precluded comparisons with those individuals with the rapid phenotype (N = 9). Therefore, the negative correlation between age and the effectiveness of NTP observed in females might be attributed to the tendency toward a NAT2 rapid phenotype in this population (75% of females). As we investigated the cells derived from surgically removed IVD tissues, the composition of the donors was dependent on the morbidity of the disc diseases that required discectomy. The frequency of female patients in the current study (38.7%) was not largely different from the frequency of female patients reported in the literature (35.2-35.6%), among whom SNPs in the THB2 and SKT genes were reported to be responsible for the susceptibility to disc diseases, such as lumbar disc herniation, in Japan [34,35]. Moreover, the median age of the donors was 56 years. Therefore, it is di cult to recruit cell donors without bias in sex and age. Second, the main components of NTP that activate the PI3-AKT pathway have not been elucidated, and there are no clues regarding whether NTP contains arylamine or a large variety of its analogs. Further constituent analyses of NTP are hence needed to identify its promoting or inhibitory active ingredients, to develop a new puri cation technique to customize NTP for IVD regeneration through the upregulation of aggrecan synthesis in the disc.

Conclusions
Overall, our study suggests an association between the promoting effect of NTP on aggrecan gene expression in NP cells and genetic polymorphisms in NAT2 in cell donors. The NAT2 intermediate acetylator phenotype signi cantly favored the effectiveness of the NTP treatment by enhancing aggrecan production in NP cells. In particular, males classi ed as having this phenotype were the most feasible population. Sex and age may also be implicated in the e cacy of NTP, although further studies including larger cohorts are needed. The above evidence suggests the potential value of NAT2 genotyping in the selection of patients that respond to NTP treatment, which represents a new application of NTP as a medicine for the restoration of deteriorated disc matrix. , and was conducted in accordance with approved protocols. Informed consent forms with written provision were completed by all patients before the donation of samples.

Consent for publication
NAT2 genotyping data and intervertebral disc samples were obtained with written informed consent. All data are anonymized and no information is traceable to any individual patient. All authors have read the manuscript and agreed to its content. This work is original and is not currently under consideration by another journal. A funder provided research grant for this study, Nippon Zoki Pharmaceutical Company Ltd. consented to publish this manuscript.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author D.S., upon reasonable requests. The individual genomic data are not publicly available due to privacy or ethical restrictions. The microarray data are available in the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/gds/?term=GSE114169. The SNP data is available on a database built by Democritus University of Thrace, http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.htm.
received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. The authors do not have any other nancial competing interests in relation to this paper.

Funding
The