2025 Volume 50 Issue 12 Pages 707-715
Asthma attacks during pregnancy can cause inadequate oxygen supply to the fetus because the mother cannot get enough oxygen into her bloodstream. Even during pregnancy, medication such as theophylline is needed to avoid asthma seizure. Theophylline is metabolized by (Cytochrome P450) CYP1A2 in the maternal liver. On the other hand, whether theophylline transferred from the mother to the fetus is metabolized in its liver depends on the degree of CYP1A2 expression, but no such information is available. In the adult liver, CYP1A2 forms zonation structures to efficiently metabolize drugs and foreign substances. In this study, we quantitatively analyzed the expression levels of mCyp1a2 mRNA and protein in the liver throughout development, from fetal and neonatal stages to adulthood, while distinguishing between males and females. In addition, to determine the timing of the mCyp1a2 zonation, analysis was performed in fetal and neonatal livers. We found that mCyp1a2 males and females express very little mCyp1a2 in the fetal liver. One week after birth, mCyp1a2 protein expression was 24% and 14% of adult expression in males and females, respectively. Furthermore, the expression level of mCyp1a2 in both sexes was almost the same as that of adults after 28 days of age. In addition, they found that the zonation of mCyp1a2 begins to form 5 days after birth. It was found that mCyp1a2 expression is very low in the fetal liver, while the liver of a 14-day-old (P14) has approximately half the drug-metabolizing capacity compared to that of an adult liver.
The incidence of asthma attacks in pregnant women with pre-existing asthma is approximately 4% (Murphy and Gibson, 2011; Schatz and Dombrowski, 2009; Tan and Thomson, 2000). Asthma attacks during pregnancy can limit oxygen supply to the fetus because the mother may struggle to get enough oxygen into her bloodstream (Schatz and Dombrowski, 2009; Osur, 2005). If the fetus experiences hypoxia, it may fail to develop properly in the uterus, which can result in fetal growth restriction. It is essential to use medication to prevent asthma attacks, even during pregnancy. In other words, the impact of an asthma attack on the mother is greater than the potential side effects on the fetus caused by the mother taking the drug. Asthmatic patients should receive treatment with theophylline and other medications, even during pregnancy. Theophylline readily crosses the placenta and reaches the fetus due to its low molecular weight (MW: 180) and high fat solubility (Walker et al., 2011; Arwood et al., 1979; Omarini et al., 1993).
Theophylline is about 90% metabolized by CYP1A2 in the adult liver and is excreted by the kidneys as a metabolite. In the words, the metabolism of theophylline is reliant on CYP1A2 expression in the liver. Pregnancy leads to changes in pharmacokinetics because of physiological alterations in the body (Abduljalil et al., 2020; Anderson, 2005, 2010; Meakin et al., 2022; Tajima et al., 2013).
Caffeine in coffee, green tea, black tea, and other popular beverages is metabolized to theophylline primarily by CYP1A2 and other pathways. This indicates that even without taking theophylline, it can easily be derived from caffeine-containing beverages. WHO recommends a daily caffeine intake of less than 200 mg for pregnant women (Gleason et al., 2021; Kukkonen et al., 2024; Rhee et al., 2015; Sasaki et al., 2017). This amount of caffeine is equivalent to consuming 3 to 4 cups of coffee daily. Caffeine is likely metabolized into theophylline in the maternal liver and subsequently transferred to the fetus, potentially without the pregnant woman's intention.
The expression of the CYP superfamily in the liver generally increases during development and decreases with age (Kwak et al., 2015; Warrington et al., 2004; Xu et al., 2019). Furthermore, the expression of many CYPs is affected by growth hormone and estrogen, leading to differences in expression patterns between females and males (Choi et al., 2013; Tsuchiya et al., 2005; Oinonen and Lindros, 1998). In the adult liver, the CYP superfamily is prominently expressed in hepatocytes near the central vein, with minimal expression around the portal vein. This expression pattern is known as zonation structure and is believed to be crucial for the efficient metabolism of drugs.
Currently, there is limited information on the expression of CYP family members responsible for metabolizing these drugs in the fetal and neonatal liver. Our research group has examined the expression levels of the Cyp3a and Cyp2c families in the liver of mice from the fetal stage through the neonatal stage to adulthood, differentiating between males and females (Hirose et al., 2016; Kawamura et al., 2022; Kitaoka et al., 2018a; Kitaoka et al., 2018b; Ochiai et al., 2016).
A study on CYP1A2 expression in the liver of human aborted fetuses found that CYP1A2 is not expressed during certain fetal stages (Sasaki et al., 2017; Sonnier and Cresteil, 1998; Walker et al., 2011).
However, the human specimens used in these studies are from pregnancies where abortion is possible (less than 22 weeks). Additionally, although CYP is influenced by growth hormone and estrogen, no analysis has been conducted to differentiate between males and females. Due to the unavailability of abortion after 22 weeks of gestation, we could not compare changes in CYP1A2 expression.
In this study, quantitative PCR and Western blotting were used in mice to analyze the changes in Cyp1a2 expression from fetal to neonatal and adult stages. Moreover, the timing of the formation of Cyp1a2 zonation structures in the liver was determined using immunohistochemical staining.
Pregnancy ICR mice and adult mice were purchased from Japan SLC, Inc. The mice were kept at room temperature (24 ± 1°C) and 55 ± 5% humidity with 12 hr of light (artificial illumination; 08:00–20:00). Food and water were available ad libitum. Each animal was used only once. The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, as adopted by the Animal Care Committee of Hoshi University. All procedures using animals were carried out according to protocols approved by the Animal Care Committee of Hoshi University.
Collection of liver samplesThree pregnant mice were used for each stage. For each pregnant mouse, the livers of six females and six males were removed. Female and male fetuses were distinguished by observing the testes and ovaries under a stereomicroscope. After extracting genomic DNA from the tails of the fetuses, we reconfirmed the discrimination of males and females by the presence or absence of the Y-chromosome-specific gene Sry gene (data not shown). The livers were instantly frozen in liquid nitrogen and stored at -80°C until preparation of the microsomal fraction. The livers of the remaining three females and three males were immersed in TRI reagent for extraction of total RNA and stored at -80°C.
Preparation of microsomal fractions from liver1 mL of crush buffer (final concentrations: sucrose 300 mM, imidazole 25 mM, EDTA 1 mM) was combined with a Protease Inhibitor Cocktail (final concentration: 1% v/v) and added to the excised liver. The mixture was homogenized on ice for 1 min using a cutter-type homogenizer. Then, centrifugation was conducted at 9,000 × g for 20 min at 4°C. After this step, 800 μL of the supernatant was collected. The collected supernatant was then ultracentrifuged (using CS120GXL, Hitachi Koki Co., Ltd., Tokyo, Japan) at 105,000 × g for 1 hr at 4°C.
After ultracentrifugation, 100 μL of RIPA buffer containing Protease Inhibitor Cocktail (final concentration: 1% (v/v)) was added to the precipitate, and the microsome fraction was sonicated for 1 min. The protein concentration of each microsomal fraction was measured using the BCA Assay Kit® (Thermo Fisher Scientific).
Genomic DNA preparation & Determining the sex of an individualAfter purification of genomic DNA from each individual as described in our previous paper, the mSry gene was specifically amplified by PCR to determine the sex of the individual.
Total RNA preparation & Synthesis of cDNATotal RNA was extracted from mouse liver using TRI Reagent. Total RNA concentration (µg/mL) and total RNA purity were calculated from absorbance measured at NanoDrop Lite (Thermo Fisher Scientific) wavelengths of 260 nm and 280 nm. The cDNA was synthesized from 1 µg of total RNA using the High-Capacity cDNA Synthesis Kit (Thermo Fisher Scientific) and diluted 20-fold in TE buffer to create a cDNA solution in TE buffer.
Quantitative PCRThe prepared cDNA TE buffer solution was utilized to amplify the target gene. A PCR reaction with SYBR Green (BIO-RAD, Hercules, California, USA) was conducted using the My iQTM single color real-time PCR detection system (Bio-Rad Laboratories, CA, USA).: denature: 95°C, 15 sec, annealing: 60°C, 15 sec, extension: 72°C, 30 sec/cycle. The primer sequences for mCyp1A2 and b-actin are shown in Table 1.
| Target | Forward Primer (5’ to 3’) | Reverse Primer (5’ to 3’) | Product size (bp) | Accesssion # |
|---|---|---|---|---|
| mCypla2 | AAG ACA ATG GCG GTC TCA TC | GAC GGT CAG AAA GCC GTG GT | 260 | NM_007818.3 |
| β-actin | GAG CGC AAG TAC TCT GTG TG | CGG ACT CAT CGT ACT CCT G | 97 | NM_007393 |
ICR pregnant mice at 15.5 of gestation were anesthetized with isoflurane, and embryo were removed by cesarean section. The neonate's liver (P5, P10) was removed by laparotomy under isoflurane anesthesia. The liver was fixed in 4% PFA at 4°C for 60 min (30 min for fetal liver). After washing with PBS (-), the samples were soaked in 10, 20, and 30% sucrose solutions at 4°C in sequence until the tissue sank in each solution. The samples were embedded in OCT compound (Sakura fine tech) and stored at -80°C. Then, they were sectioned into 10 µm slices using cryostat (Leica CM1850) and placed on coated-slide glass (MAS-01, MATSUNAMI). Once the sections were sufficiently dried, they were stored at -80°C as frozen sections until use.
Immunostaining with anti-CYP1A2 antibodies (Dilution 1:100, proteintech:9936-1-AP) was performed in the same manner as previously reported for CYP2C and CYP3A. The immunostained sections were detected using BZ-X800 microscope (Keyence).
Quantification of CYP1A2 protein expression by western blottingSupernatant was collected, and the protein content was determined by the BCA reagent. Protein separation and immunoblot analysis were performed with Sally Sue Simple Western System according to the manufacturer’s instructions (ProteinSimple™). In brief, samples were diluted to adjust protein concentration to 1 μg/μL in 5 μL with sample buffer and further diluted by adding 1.25 μL of the 5× master mix (ProteinSimple) to a final concentration of 1× sample buffer, 1× fluorescent molecular weight markers, and 40 mM DTT and then heated at 95 °C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies (Anti-CYP1A2, proteintech:9936-1-AP -rabbbit antibody/Antibody DiluentII 1:50), secondary antibodies (rabbit IgG-HRP, proteinSimple), and chemiluminescent substrate were dispensed into designated wells in the manufacturer-provided microplate. After plate loading (Capillary Cartridges, Jess/Wes Separation 12-230 kDa), the separation electrophoresis and immunodetection steps took place in the capillary system and were fully automated. Simple Western analysis was carried out at room temperature, and instrument default settings were used. The data were analyzed with inbuilt Compass software ver 5.0.1 (ProteinSimple).
StatisticsThe experimental values are presented as the mean value ± the standard deviation.
The Smirnov-Grubbs outliers test was used to calculate any outliers. Calculation and graphical exploration were performed using GraphPad Prism software (Prism ver.10) .
The expression level of mCyp1a2 mRNA in the liver of mice was analyzed at each developmental stage using quantitative PCR (Fig. 1AB).
In the fetal liver, mCyp1a2 expression was found to be very low in both sexes, constituting less than 5% of that observed in adults (P56). At postnatal day 7 (P7), mCyp1a2 mRNA expression in the liver was less than 10% of that seen in adults (P56) for both males and females (Fig. 1A).
The mRNA expression level of Cyp1a2 during developing liver. Livers were harvested from each developmental stage (E13.5, E15.5, E17.5, P1, P3, P7, P14, P28, P42, P56) of mice and total RNA was extracted. Then, the mRNA expression levels of mCyp1a2 was analyzed by quantitative PCR. Expression levels at each time point were expressed as 100% of Cyp1a2 expression in adult livers at postnatal day 56 (P56). Results are presented as mean ± standard deviation (n =5). (A) Analysis of mCyp1a2 mRNA expression in liver from embryonic day 13.5 to postnatal day 7. (B) An analysis of mCyp1a2 mRNA expression in the liver from embryonic day 13.5 to adulthood (postnatal day 56).
Further analysis of relative expression levels from fetal to in vivo showed a gradual increase in Cyp1a2 mRNA expression in males from P14 to P56. In the liver of female P28, Cyp1a2 mRNA expression was approximately 1.5 times higher than that of P56, indicating a significant increase (Fig. 1B).
Quantitative analysis of mCyp1a2 protein expression in the liver during embryonic, neonatal, and adult stagesIt was found that CYP1A2 mRNA is rarely expressed in the fetal stage. Therefore, to confirm the translation from mRNA to protein, we analyzed the expression level of CYP1A2 in the liver of mice from fetal stage to each developmental stage by quantitative Western blotting (Fig. 2).
The protein expression level of Cyp1a2 during developing liver. (A) JESS automated the capillary western blotting of liver microsomes (1 µg protein /lane) using antibodies specific to CYP1A2. This experiment was conducted three times independently, and representative images are shown. After the capillary western blotting images were acquired, the density of each band was quantified by software (Compass soft ware ver.5.0.1). Results are presented as mean ± standard deviation (n = 3). (B) The expression levels of Cyp1a2 in the liver from the fetal period (E13.5) to the neonatal period (P7) were quantified based on the expression levels at P56. (C) The expression levels of Cyp1a2 in the liver were quantified from the fetal stage to adulthood, using the expression levels at P56 as a reference.
Microsomal fractions from the livers of fetal, neonatal, and adult rats were analyzed for mCyp1A2 expression using quantitative Western blotting (Fig. 2). In the fetal liver, mCyp1a2 was expressed at less than 3% of its adult levels in both females and males (P56) (Fig. 2C). At 7 days of age (P7), Cyp1a2 expression in livers was 23.6% for males and 14% for females. In livers on postnatal day 28 (P28), expression levels in both males and females were found to be comparable to those in adult livers (Fig. 2C).
These results indicate that livers at 28 days of age express CYP1A2 at levels comparable to adults (P56).
Determining when mCyp1a2 zonation develops in the liverThe zonation structure of cytochrome P450 enzymes (CYP) in the liver is crucial for effective drug metabolism. We prepared liver tissue sections and examined them using immunohistochemical staining with anti-CYP1A2 antibodies to identify the formation timing of mCyp1a2 zonation structures. At E15.5 days, the liver of both males and females showed no zonation structure (data not shown). The zonation structure of mCyp1a2 in the liver gradually begins to form at P5 in both males and females (Fig. 3A, 3B). Additionally, it was observed that a distinct Cyp1a2 zonation structure developed by 10 weeks after birth (Fig. 3C, 3D).
Identification of the time of formation of Cyp1a2 zonation in the liver. The livers of mice at each stage (P5, 10 weeks) were removed, fixed in 4% PFA, thinly sliced in a cryostat, and immunohistochemically stained with anti-CYP1A2 antibody. Images A and B display the livers of male and female newborns (P5) that were immunostained with an anti-Cyp1a2 antibody. A' and B' are magnified views of A and B, respectively. Arrows indicate cells that have begun to express Cyp1a2, albeit weakly. Images C and D present the results for the livers of adult males and females, both at 10 weeks of age. Dashed circles represent the zonation structure.
In mammals, the CYP1A subfamily of drug-metabolizing enzymes includes CYP1A1 and CYP1A2. CYP1A1 is primarily expressed in tissues outside the liver, whereas CYP1A2 plays a significant role in the metabolism of drugs and foreign substances within the liver and small intestine. However, most findings related to CYP1A2 are based on studies of adult livers. It remains unclear whether theophylline transferred from mother to fetus is metabolized by CYP1A2 in the fetal liver. We analyzed whether theophylline transferred from the mother to the fetus can be metabolized in the fetal liver, using mCyp1a2 expression levels as an indicator.
In this paper, we demonstrated that mCyp1a2 expression in the fetal liver (E13.5-E17.5) is significantly lower in both males and females compared to adults, as analyzed at the mRNA and protein levels. It shows that the protein expression of mCyp1A2 in the liver increases dramatically between postnatal days 7 and 14 (Fig. 2). Consistent with previous reports, this sharp induction coincides with hepatic maturation and is thought to reflect developmental regulation by nuclear receptors such as AhR, as well as hormonal and nutritional influences after birth (Cui et al., 2012; Cui et al., 2009; Hart et al., 2009). These findings suggest that mCyp1a2 expression is under multi-layered control during liver development, and that the dramatic postnatal increase likely represents a coordinated transition in hepatic function.
Although the developmental patterns of mCyp1a2 mRNA and protein expression generally showed similar trends, some discrepancies were observed at specific stages such as P7 and P28. At P28, for instance, females exhibited higher mRNA expression than males, whereas the protein levels were nearly identical. At P7, the relative levels even appeared reversed between mRNA and protein. Such divergence between transcript and protein abundance has been widely reported and is considered a common feature of gene regulation (Liu et al., 2016). These differences may arise from multiple regulatory layers, including post-transcriptional processes such as mRNA stability and regulation by RNA-binding proteins, translational control, and differences in protein degradation rates (Vogel and Marcotte, 2012). In addition, methodological differences in sensitivity and dynamic range between qPCR and Western blotting may accentuate apparent mismatches. Taken together, these findings suggest that mCyp1a2 expression is subject to multi-layered regulation during development, and that mRNA levels alone do not necessarily predict protein abundance.
We have shown that a zonation structure for efficient drug metabolism begins to develop gradually starting from the fifth day after birth (Fig. 3). Analyzing mRNA expression levels with PCR and protein expression levels with Western blotting provides average results due to the bulk analysis of homogenized liver tissue. In other words, when expressing proteins that form zonation structures, such as cytochrome P450, there is a risk of overlooking them if they are only expressed in some liver cells. In fact, we have demonstrated that Cyp3a and Cyp2c family genes are expressed in some fetal liver cells. Therefore, we analyzed Cyp1a2 in fetal liver using immunohistochemical staining. However, unlike the Cyp3a and Cyp2c families, Cyp1a2 expression was not detected in the fetal liver.
This research indicated that CYP1A2 expression is notably low in the livers of mouse fetuses. Therefore, it was considered that when drugs that are substrates of CYP1A2, such as theophylline, are transferred from the mother to the fetus, they cannot be metabolized sufficiently. Furthermore, it was found that the expression level of Cyp1A2 in the liver of newborns one week after birth was approximately 20% that of adults. The dosage of theophylline for newborns is about half that for adults, according to the package insert (Anderson and Lynn, 2009). Based on our results, it is thought that the dosage of theophylline administered to newborns should be reduced to approximately one-fifth of the adult dosage. In the future, it will be essential to administer CYP1A2 substrate drugs, such as theophylline, to mice during pregnancy and the neonatal period. This will help clarify the effects of these drugs on fetuses and newborns, as well as analyze changes in CYP1A2 expression in the liver.
The findings of this study are applicable to all CYP1A2 substrate drugs, and we believe they will enhance drug treatment by considering the liver's metabolic capacity in fetuses and newborns.
Cyp1a2 expression is regulated not only by developmental processes but also by inducible pathways mediated by the AhR (Nebert et al., 1993). Environmental factors such as dioxins and polycyclic aromatic hydrocarbons, including those from tobacco smoke, can strongly influence Cyp1a2 expression (Hankinson, 2016, Molden and Spigset, 2009). In our present mouse experimental system, we did not introduce exogenous AhR ligands; therefore, the observed developmental expression patterns primarily reflect endogenous regulation. Nevertheless, environmental exposure in vivo could affect hepatic zonation and potentially alter Cyp1a2 expression from the fetal stage onward. We cannot exclude these possibilities, and we consider them an important subject for future investigation.
We thank all members of the Department of Pharmacokinetics, Hoshi University for their help and advice.
FundingNo funding was provided for the work.
Conflict of interestThe authors declare that there is no conflict of interest.
Data availabilityThe data in this study are included in the article/supplementary materials. Contact the corresponding author(s) directly to request the underlying data.
Author contributionsConceptualization: Wataru Ochiai
Investigation: Mako Ichikawa, Miyuki Yagi, Kurumi Miyazawa, Hiromasa Muto
Supervision: Hiromasa Kato
Visualization: Wataru Ochiai
Writing – original draft: Mako Ichikawa
Writing – review & editing: Wataru Ochiai, Hiromasa Kato
Ethical approval and consent to participateNot applicable.
Patient consent for publicationNot applicable.
