Sex differences in CYP450-based sodium dehydroacetate metabolism and its metabolites in rats

Optimization of incubation conditions for liver microsomes

We first optimized the incubation condition of a liver microsome system in vitro. Different concentrations of liver microsomes (0.25 or 0.1 mg/L) were incubated with DHA-Na (50, 100, and 167 mg/L) for different periods. According to the DHA-Na reaction degrees (Fig. S1), we utilized the 0.1 mg/L microsome system with 5.0 mg/L DHA-Na and a treatment period of 3 hours for subsequent experiments.

Inhibition of CYP450 sub-enzymes by specific inhibitors in vitro

To verify the effects of CYP450 sub-enzyme inhibitors under experimental conditions, we detected the content of relevant probe substrates after co-incubation with specific inhibitors and substrates separately through High-performance liquid chromatography (HPLC). The chromatograms for all five probe substrates are illustrated in Fig. S2. Moreover, the ranges of their standard curve equations, recovery rates, precision rates, Limits of detection (LODs), and Limits of quantification (LOQs) were presented in Table 1.

Compared with the control groups, the levels of five specific probe substrates were notably lower in the negative control male and female liver microsome groups. Moreover, the levels of five probe substrates were lower in the male liver microsome groups than in the female liver microsome groups (Fig. 1A–E). 25 μM specific inhibitors treatment considerably increased the contents of probe substrates. Specifically, metabolic rates for phenacetin, dapsone, dextromethorphan, omeprazole, and chlorzoxazone were reduced respectively by 38.21%, 42.75%, 50.52%, 9.66%, and 47.50% in the male groups and by 42.51%, 65.71%, 72.38%, 80.22%, and 70.71% in the female groups. These results indicated that at 25 μM, the specific sub-enzyme inhibitors effectively inhibited their corresponding sub-enzymes; however, the degree of inhibition differed between sexes.

Identification of CYP450 sub-enzymes involved in DHA-Na metabolism in vitro

To identify CYP450 sub-enzymes involved in DHA-Na metabolism in vitro, we performed HPLC analysis of DHA-Na concentrations after coincubation of the liver microsome systems with 5 mg/L DHA-Na and specific sub-enzyme inhibitors for 3 h. As shown in Fig. 2A, the male and female groups treated with 5–50 μM furafylline (a CYP1A2 inhibitor) exhibited higher DHA-Na concentrations than the control groups; moreover, the differences among the male groups treated with 40 and 50 μM furafylline and among the female groups treated with 20–50 μM furafylline were significant. Furthermore, DHA-Na concentrations were higher in the female groups than in the male groups after treatment with 5–50 μM furafylline. In contrast, DHA-Na concentrations in the male group treated with 10 μM furafylline were similar to those in the female control group. As shown in Fig. 2B, DHA-Na concentrations in both female and male liver microsomes increased after treatment with 10–50 μM ketoconazole (a CYP3A2 inhibitor). Moreover, DHA-Na concentrations were higher in all the female groups than in the male groups treated with 10–50 μM ketoconazole. In contrast, DHA-Na concentrations in the male group treated with 40 μM ketoconazole were similar to those in the female control group. As shown in Fig. 2C, DHA-Na concentrations were considerably higher in the male group treated with 50 μM quinidine and female groups treated with 20–50 μM quinidine than in the control groups. As illustrated in Fig. 2D, E, CYP2C11 and CYP2E1 inhibition with ticlopidine and clomethiazole did not lead to a significant change in DHA-Na concentrations.

A–E Concentrations of DHA-Na after coincubation with DHA-Na and 5–50 μM furafylline (A), ketoconazole (B), quinidine (C), ticlopidine (D), or chlormethiazole (E). *P < 0.05, **P < 0.01, compared with control group. ^#P < 0.05, compared with male group.

Table 2 presents DHA-Na metabolism inhibition rates in liver microsomes after treatment with 20–50 μM furafylline, quinidine, quinidine, ticlopidine, or clomethiazole respectively. Our results indicated that furafylline, quinidine, and quinidine reduced DHA-Na metabolism respectively by 12.23% ± 1.96% to 23.48% ± 3.61%, 5.70% ± 0.74% to 15.40% ± 1.91%, and 4.41% ± 0.62% to 6.81% ± 0.82% in the male groups and by 16.52% ± 3.84% to 33.93% ± 3.83%, 7.62% ± 1.31% to 20.77% ± 3.72%, and 6.72% ± 1.26% to 12.42% ± 2.64% in the female groups. In all groups, ticlopidine and clomethiazole led to DHA-Na metabolism inhibition rates of <4%. In summary, CYP1A2, CYP3A2, and CYP2D1 were noted to be involved in DHA-Na metabolism in liver microsomes, with CYP1A2 and CYP3A2 playing the key roles.

Inhibition of CYP450 sub-enzymes by specific inhibitors in vivo

Table 3 shows the standard curve equations, recovery rates, precision rates, LODs, and LOQs of all five probe substrates in vivo. The ranges of all LODs and LOQs were 0.0125–0.0500 and 0.0333–0.0750 mg/L, respectively.

To evaluate the inhibitory effects of specific inhibitors on CYP450 sub-enzymes in vivo, we administered rats with one of the inhibitors over 3 days. Next, the serum concentrations of probe drugs were analyzed through HPLC 1, 2, 4, and 7 h after pore drug administration (Fig. S3). As shown in Fig. 3A–E, the serum concentrations of phenacetin 1 h after administration, as well as those of dapsone and omeprazole 1–7 h after administration, were significantly lower in the male rats than in the female rats. All rats, regardless of their sex, demonstrated a considerable increase in the serum concentration of probe substrates after specific inhibitor treatments (Fig. 3A–E), indicating the effectiveness of the inhibitors.

Concentration–time curves for probe substrates in rats after coadministration of (A) 5 mg/kg furafylline and 10 mg/kg phenacetin, (B) 5 mg/kg dapsone and 25 mg/kg ketoconazole, (C) 5 mg/kg dextromethorphan and 10 mg/kg quinidine, (D) 10 mg/kg omeprazole and 10 mg/kg ticlopidine co-administration. E 5 mg/kg chlorzoxazone and 25 mg/kg chlormethiazole.

Identification of CYP450 sub-enzymes involved in DHA-Na metabolism in vivo

To further elucidate DHA-Na metabolism in vivo, we detected serum DHA-Na concentrations in rats treated with specific CYP450 subenzyme inhibitors for 3 days. Figure 4 illustrates serum DHA-Na concentration–time curves after CYP450 subenzyme inhibitor administration. Treatment with 5 mg/kg furafylline, 25 mg/kg ketoconazole, or 10 mg/kg quinidine increased serum DHA-Na concentrations in the male and female rats (Fig. 4A–C). However, ticlopidine or clomethiazole treatment led to no obvious changes in serum DHA-Na concentrations (Fig. 4D, E). Furthermore, regardless of CYP450 subenzyme inhibitor treatment, serum DHA-Na concentrations remained higher in all female rats than in all male rats (Fig. 4).

Concentration–time curves for DHA-Na after coadministration of 200 mg/kg DHA-Na and (A) 5 mg/kg furafylline, (B) 25 mg/kg ketoconazole, (C) 10 mg/kg quinidine, (D) 10 mg/kg ticlopidine, and (E) 25 mg/kg chlormethiazole.

Table 4 presents the pharmacokinetic parameters of DHA-Na in rats after specific inhibitor administration. In the control groups, the half-life (t1/2), maximum concentration (C_max), area under the curve from 0 to the last measurable timepoint (AUC _(0-t)), area under the curve from 0 to infinity (AUC _(0-∞)), and mean residence time (MRT) were higher in the female rats than in the male rats. After furafylline treatment, the t1/2, Cmax, AUC _(0-t), AUC _(0-∞), and MRT of DHA-Na significantly increased in all treatment groups; they were higher in the female group than in the male group. Moreover, the clearance (CL) and volume of distribution (Vd) of DHA-Na decreased only in the female group. In addition, in the male group, the CL was lower than that in the control group but higher than that in the female group. After ketoconazole treatment, the C_max, AUC_(0-t), and AUC_(0-∞) of DHA-Na increased considerably in all treatment groups; moreover, the t_1/2 and MRT of DHA-Na increased considerably in only the female group, and the Vd and CL of DHA-Na increased considerably in only the male group. In addition, the C_max, AUC_(0-t), AUC_(0-∞), t_1/2, and MRT of DHA-Na were higher in the female group than in the male group. After quinidine treatment, the t_1/2, C_max, AUC_(0-t), and AUC_(0-∞) of DHA-Na increased, but its Vd and CL considerably decreased in all treatment groups. Moreover, the MRT of DHA-Na increased substantially in the male group. In addition, compared with the female group, the male group demonstrated higher AUC_(0-t) and AUC_(0-∞) but lower CL of DHA-Na. After ticlopidine treatment, the female and male groups demonstrated higher C_max of DHA-Na, whereas only the male group demonstrated higher MRT of DHA-Na. Finally, clomethiazole treatment led to no obvious changes in the treatment and control groups. Nevertheless, the C_max, AUC_(0-t), AUC_(0-∞), and MRT of DHA-Na were higher in the female group than in the male group. Taken together, these results confirmed that CYP1A2, CYP3A2, and CYP2D1 are involved mainly in DHA-Na metabolism.

Sex differences in Cyp450 expression

To investigate the reasons underlying the sex differences in the activities of sub-enzymes, we analyzed the mRNA expression of the genes encoding these sub-enzymes in male and female rats. As shown in Fig. 5A, B, Cyp1a2, Cyp3a2, Cyp2d1, and Cyp2c11 mRNA expression was significantly lower in female rats than in male rats; however, no significant difference was noted in Cyp2e1 mRNA expression between male and female rats.

A RT-PCR products; lanes 1–3 and 4–6 contain male and female rat samples, respectively. B Relative expression of all five CYP450 sub-enzymes. *P < 0.05, **P < 0.01.

DHA-Na metabolites in rats

Two peaks, M1 and M2, were observed in the DHA-Na chromatograms of rat serum at approximately 3 min. As illustrated in Fig. S4, the peak heights of both M1 and M2 increased over time. Although no relevant data are available, we speculated that these peaks corresponded to DHA-Na metabolites. To identify these two substances, rat sera treated with DHA-Na were assessed through liquid chromatography–mass spectrometry (LC/MS). The LC/MS total ion chromatograms of DHA-Na and its metabolites are presented in Fig. 6A–D. In particular, the accurate mass spectra of DHA-Na and its metabolites were recorded and examined for characteristic product ions. As depicted in Fig. 6C, D, product ions m/z 167.0354 were identified as DHA-Na, and the product ions m/z 183.0288 and 139.0401 were defined as M1 and M2, respectively. To elucidate M1 and M2 structures, we performed secondary mass spectrometry. As shown in Fig. 7A–C, the secondary mass spectra demonstrated that product ions m/z 107.0482 and 83.0132 were identified as M1 fragment ions. Based on the tandem mass spectrometry database, the structure of M1 was elucidated to correspond to C₈H₇O₅, whereas the structure of M2, a monoderivative product of M1, was elucidated to correspond to C₆H₅O₃N (Fig. 7B). As shown in Fig. 7C, we identified N1 (m/z 107.0482) to be a fragment ion formed via the elimination of one acetyl and one ketone group from M1 and N2 (m/z = 83.0132) to be formed as a fragment ion formed via the elimination of oxygen and one methyl group from the N1 pyran ring.

A, B Total ion current (A) and selective ion flow (B) patterns of DHA-Na and its metabolites M1 and M2. C Internal mass spectrograms of M1 (2.61–2.72 min; C) and M2 (1.85–1.94 min; D).

A, B Mass spectra of DHA-Na (A) and its metabolites M1 and M2 (B). C Secondary mass spectra of metabolite M1.