The animals used in this study were the Sprague Dawley strain of Rattus norvegicus. All animal experimentation was approved by the University of Melbourne Animal Ethics Committee (Ethics Permission AEC: 1714344.1) and conducted in compliance with Australian National Health and Medical Research Guidelines. All animals were assessed as healthy prior to commencement of experiments. Animals were monitored prior to and following every injection ensuring there was no abnormalities in weight (>10%), appearance (fur) or behaviour (vocalisation, respiration, movements). All efforts were made to ameliorate any suffering of animals. They were handled by experienced researchers in such a way as to minimise stress prior to being anaesthetised.

These were supplied by the University of Melbourne Biological Research Facility and subjected to a 12 hour light/dark cycle with ad libitum access to food (dry pellets of a fixed formulation diet for laboratory rats and mice fortified with vitamins and minerals to meet the requirements of breeding animals after the diet is autoclaved or irradiated, supplied by Speciality Feeds, Western Australia) and water. Animals were housed in groups of 2–4 (adult) per cage (25cm x 35cm x 25cm on Breeders Choice paper bedding, made from 99% recycled paper; it is biodegradable with no added chemicals). Age groups investigated (at treatment completion) were embryonic day 19 (E19) pups of both sexes and dams, which were all primigravida 350–400g body weight) and non-pregnant female adults (175–230g body weight). E19 was chosen because this is a stage of development when adequate volumes of blood and cerebrospinal fluid (CSF) can be obtained for analysis from fetal rats without pooling ( Dziegielewska et al., 1981) and individual pups can be injected intraperitoneally while still inside the uterine horn and kept viable for periods of time. Animal numbers were based on previous experience of such experiments and were the minimum number required to detect a significant difference between groups at p <0.05. Animals were selected for treatment groups to ensure weights were statistically similar between direct comparisons. Where possible, equal numbers of male and female fetuses were used. Animals on gestational day E19 were allocated to experiments by animal house staff, who had no knowledge of the particular experiments to be performed. The experimenters had no role in the selection of the animals, thus avoiding selection bias. The numbers (n) of animals used for each experiment are indicated in the relevant Methods or Results section and where appropriate in legends. Two litters in the sucrose permeability studies were excluded from the study. One mother died under anaesthesia. In the other case the fetuses were observed to be in poor physiological state, which would have affected the results.

Paracetamol (acetaminophen ≥99.0%, Sigma-Aldrich) was applied either at a high dose of 15mg/kg (higher limit in the range used clinically, Australian Medicines Handbook, 2019 and Koehn et al., 2019b) or a dose in the lower clinical range of 3.75mg/kg. Paracetamol was dissolved in sterile 0.9% sodium chloride solution for injection. For passive permeability experiments [U- ¹⁴C]-labelled sucrose (Amersham International, CFB146) was injected in sterile 0.9% sodium chloride solution. Details are described in our previous study ( Koehn et al., 2019b). Estimates of protein (AFP) permeability were obtained from western blot analysis of fetal and maternal plasma, as described below.

All experiments took place between 09.00 and 15.00h. Placentas and fetal brains from dams subjected to three treatment regimes were analysed in this study (n=4 for each tissue from each dam).

In experiments (i) and (ii), 30 minutes after the last injection of the drug the tissue samples (placentas, fetal brains) were collected (n=4 for each dam).

For RNA-Seq analysis, placental tissue was sampled as a cross section of the chorio-allantoic placental disc, following removal of the externally attached umbilical and maternal circulatory vessels. Brain samples of the cortex were dissected out as described before ( Koehn et al., 2019a). Samples were collected under RNase free conditions and immediately frozen in liquid nitrogen and transferred to -80 °C for storage. RNA extraction was completed using the RNeasy Plus Mini Kits and QIAshredder (Qiagen, catalogue number 74134) for placenta and using the RNeasy Plus Micro Kits (Qiagen, catalogue number 74004) for fetal cortex, following manufacturers specifications. RNA quantity and purity were determined using a NanoDrop ND-1000 UV-VIS spectrophotometer (Thermo Scientific).

RNA samples were transported on dry ice to the Australian Genome Research Facility (AGRF) in Melbourne for Illumina, next-generation sequencing. Runs were 100bp single reads, providing raw FASTAq data. Data were processed using the Galaxy platform and their online software packages ( Afgan et al., 2018). Default parameters were used unless directly specified. Alignment was conducted using HISAT2 (Galaxy version 2.1.0) using the reference genome for rat (rn6; accession number GCA_000001895.4) and the reverse strand setting. For transcript quantification and differential expression analysis, three different methods were employed. In the first, pathway transcripts were assembled with cufflinks (Galaxy version 2.2.1.2) using the reference annotation for rat RefGene (genome) obtained from UCSC Main. Relevant data were passed through Cuffmerge (Galaxy version 2.2.1.1) and analysed for differential expression between groups of interests using Cuffdiff (Galaxy version 2.2.1.5). For the second and third pathway, counts were aligned using HTSeq-counts (Galaxy version 0.9.1) using the reverse strand setting. Generate Count Matrix (Galaxy Australia version 1.0) produced a matrix form of the data, which were then fed through either DEseq2 (Galaxy version 2.11.40.6) or EdgeR (likelihood ratio; Galaxy version 3.24.1) to receive differential expression analysis between treatment groups. Statistically different expression levels between relevant treatment groups were selected if present in two of the three datasets above the statistical threshold of p <0.05 for the adjusted P value of Cuffdiff (Padj), DEseq2 (q value) or EdgeR (FDR). This method of statistical selection minimizes the known false positives and false negatives that can be obtained due to the analysis pathway selected, ensuring all results can be found between multiple pipelines (see Seyednasrollah et al., 2016; Soneson & Delorenzi, 2013). Gene synonym names were produced via bioDBnet ( Mudunuri et al., 2009). Pathway analysis was conducted using DAVID Bioinformatics Resources (version 6.8), with benjamini false discovery rate correction ( Huang et al., 2009a; Huang et al., 2009b).

IL1β cytokine concentrations in rat plasma were determined using ELISA specific for rat IL1β (R&D systems, Quantikine kit, catalogue number RDSRLB00, monoclonal mouse anti-rat IL1β) following the manufacturer’s protocol. Plasma samples were diluted 1:2 and 50μL of each sample was added to the same volume of assay diluent. Standard dilutions were assayed in duplicate. The plate was incubated at room temperature for two hours, then washed extensively. 100μl of rat IL1β conjugate was added and incubated for a further two hours. After additional thorough washes, the plate was incubated for 30minutes in 100μL of substrate solution then developed with 100μl of stop solution. Plates were read using a FlexStation 3 Multimode Microplate Reader (wavelength 450nm, using 570nm to correct for any optical imperfections in the plate) within 30 minutes of the addition of the stop solution. Cytokine concentrations were determined by comparison with the standard curve produced from each run.

All permeability experiments were conducted on E19 dams and fetuses. Two chronic paracetamol treatment regimes were used. Time-mated E15 pregnant dams were injected i.p. twice daily with either a 15mg/kg (referred to as “high”) or 3.75mg/kg (referred to as “low”) dose of paracetamol (dissolved in sterile 0.9% sodium chloride solution) over four days (“chronic” experiments). On the 5 ^th day, at E19, these were compared to age-matched animals that were not treated (controls). Numbers (n) of individual experiments are indicated below and are included in the legends of corresponding figures in the Results section.

¹⁴C-sucrose permeability. Animals were treated either with a “low” dose (3.75mg/kg) or “high” dose (15mg/kg) of paracetamol over four days starting at E15 following the same protocol as above. On the 5 ^th day the pregnant dams (E19) were anaesthetised i.p. with 25% w/v urethane, (Sigma, 1ml per 100g body weight) and placed supine on a 35°C heating plate and an endotracheal cannula inserted prior to sampling. Left femoral artery and vein were cannulated. All injections were by slow infusion to the femoral vein; the cannula was flushed with 2ml of heparinized (Hospira Inc, 5000 units per ml) saline. Maternal blood samples were taken from the femoral artery; blood volume was maintained by intraarterial injection of equivalent volumes of 1ml heparinized sodium chloride solution. Blood (right cardiac ventricle), CSF (cisterna magna) and brains (cortex) were sampled from each fetus. Sampling was concluded when the state of the placental circulation (normal condition: umbilical veins pink with oxygenated blood) was deemed insufficient, usually around 90 minutes (see Koehn et al., 2019b for details). CSF samples were examined microscopically for traces of red blood cells and discarded if contaminated ( Habgood et al., 1992). Maternal blood was also collected at the end of the experiment. Blood samples were centrifuged (5000rpm, five minutes). Plasma supernatant was removed and stored at -20°C until used.

Two sets of permeability experiments were conducted:

Permeability of a fetal specific protein, AFP- western blotting. Levels of AFP in both the maternal blood samples and in fetal samples obtained from experiments of paracetamol treated dams as described above, were estimated using western blotting and antibodies to human AFP (DAKO).

All plasma samples were diluted 10-fold in isotonic saline (0.9%) prior to sample preparation. Samples were run using a total of 9µL of dam and 2µL of diluted fetal sera, denatured in 4x sample buffer (62.2 mM Tris, 5% (v/v) glycerol, 2% (w/v) SDS, and 0.0025% (w/v) bromophenol blue), heated to 95°C for five minutes and centrifuged briefly to remove potential particular matter. 12µL of each sample was loaded onto a 4–12% NuPAGE Novex Bis-Tris Midi gel (Life Technologies) and proteins were resolved at 200V for 40 minutes immersed in MES SDS running buffer (Life Technologies). Gel-resolved proteins were transferred onto PVDF membranes using iBlot gel transfer stacks (iBlot 2; Life Technologies) as per manufacturer's instructions. Membranes were incubated for one hour at room temperature in PBS-T blocking buffer (PBS supplemented with 0.05% (v/v) Tween-20 [Chemsupply]) and 5% (w/v) skim milk powder. Membrane was incubated with AFP primary antibody (AFP, rabbit polyclonal, 1:1000, DAKO, catalogue number A0008, RRID AB_2650473) diluted in the blocking buffer and incubated overnight at 4°C. After three PBS-T washes, the membrane was incubated for two hours at room temperature in horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling; 1:5000, catalogue number 7074) secondary antibody. Immunoreactive protein bands were visualised by adding 1mL of Enhanced Chemiluminescence mixture (ECL Advance, GE Healthcare) onto membranes and detecting luminescence using a FujiFilm LAS-3000 imager at three and 75 second exposures. Densitometric quantitation of immunoreactivity was performed using ImageJ 2-bit, v1.46 run on OSx 10.14 Mojave on 8-bit TIFF file images. All samples that were directly compared were run on the same gel. Serum from an age-matched non-pregnant female was used as a negative control, while an E19 pregnant dam that was not injected with paracetamol was used as a positive control. Both samples were included on every gel.

Blood-brain barrier permeability in the fetus was estimated using (i) radioactive sucrose as an example of a small molecular marker permeability and (ii) plasma protein (immunohistochemistry), as an example of a large molecular marker permeability ( Habgood et al., 1993; Johansson et al., 2008; Stolp et al., 2005b). Fetal blood, CSF and brain samples were obtained from the same placental permeability experiments described above.

¹⁴C-sucrose permeability. For estimation of transfer from mother to fetus, pregnant animals treated with paracetamol (as above) were anaesthetized i.p. with urethane. Starting at 30 minutes after the last maternal injection, embryos were individually extracted. For estimation of transfer from fetal blood to fetal brain and CSF, the fetuses were exposed and injected i.p. with ¹⁴C-sucrose.

In both types of experiment fetal blood and CSF were sampled as described previously ( Koehn et al., 2019b). Fetal brain samples were taken by opening the cerebral hemispheres to expose the lateral ventricles and a sample of the parietal cortex was removed, taking care to avoid the choroid plexuses. Brain or CSF to plasma ratios of ¹⁴C-sucrose radioactive counts were used as an estimate of the transfer of sucrose across the blood brain barriers. These were calculated using the equation:

B r a i n o r C S F t r a n s f e r = B r a i n o r C S F D P M / μ l P l a s m a D P M / μ l × 100 %

Treatment groups investigated were control, no paracetamol (n=13), chronic low dose (3.75mg/kg, n=11) and chronic high dose (15mg/kg, n=11) in fetuses that were injected directly. In experiments in which the ¹⁴C-sucrose was injected into the treated mothers, numbers of pups used were control (n=8), acute (n=10) and chronic high dose (n=6).

Immunohistochemistry. Individual fetal brains were fixed in Bouin’s fixative for 24–48h then dehydrated through graded alcohols, cleared in chloroform and embedded into paraffin wax blocks. These blocks were cut serially into coronal 5µm sections (Leica microtome). Selected sections were heated for 30 minutes (60°C) then washed twice with Histolene (Fronine) for 10 minutes, then five minutes. The sections were rehydrated through graded alcohols for five minutes each (100%, 100%, 95%, 70%) and washed in phosphate buffered saline with 0.2% Tween20 for five minutes. Peroxidase and protein blockers (DAKO) were added to sections and incubated at room temperature for two hours each to block non-specific binding. The primary antibody, plasma protein (anti-rat whole serum, SIGMA, catalogue number R5129, rabbit polyclonal) diluted 1:3000 in a blocker (0.5% fish gelatine and PBS + Tween20), was applied to the slides and incubated overnight at 4°C. After three washes of PBS + Tween20 for five minutes each, the secondary (swine anti-rabbit, DAKO, catalogue number Z0196, polyclonal) and tertiary antibodies (rabbit PAP, SIGMA, catalogue number P1291) both diluted 1:200 were each added and incubated for two hours at room temperature with washes between incubations. Sections were developed with DAB (Diaminobenzidine) using DAKO DAB+ kit (catalogue number K3468) according to manufacturer’s directions and washed in running water for five minutes. Sections were dehydrated through a series of graded alcohols (70%, 95% for five minutes, then 100% for 10 minutes), then 3x five minutes in histolene washes. Slides were then mounted using DPX mounting medium (Fronine). Stained sections were examined under a compound microscope (Olympus, BX50) fitted with a digital camera (Olympus DP70). One control slide was included with every round of immunostaining and had the primary antibody omitted but was otherwise treated in the same way. These were always blank. A total of 11 brains with at least two brains per treatment group were prepared and serially sectioned and mounted on glass slides. Each slide contained 6–8 sections, every 10th slide was stained with haematoxylin and eosin for general morphology. One or two adjacent slides per brain were immunostained for plasma protein from comparable brain regions.

Plasma (10μL), CSF and every injectate (1μL of 1:10 dilution) were weighed and transferred into scintillation vials. In all experiments the radioactivity in the injectate was measured to confirm the uniformity of the injected material. Soluene350 (0.5ml, PerkinElmer) was added to the brain samples and incubated overnight at 36°C. Prior to measurement, two drops of glacial acetic acid (Sigma) were added to brain vials to neutralize the strongly alkaline Soluene350. All samples were mixed with 5ml of scintillation fluid (Emulsifier-safe, PerkinElmer) and measured on the liquid scintillation counter (Tri-Carb 4910 TR, PerkinElmer). Counting was conducted in disintegrations per minute (DPM) for five minutes each with luminescence correction on. Vials containing control, non-radioactive tissues processed identically were also counted simultaneously to establish background counts (which were subtracted from all radioactive samples). Counts were normalized to the sample weight and expressed as DPM per µL or µg of sample. Results are described as concentration ratios, defined as a % of the counts (per µL or µg) in the compartment of interest (brain, CSF, maternal or fetal plasma) divided by the counts (per µL) in the plasma compartment of comparison (see also Koehn et al., 2019b).

RNA-Seq data analysis is detailed above, with significance set at p <0.05. For all other experimentation, statistical differences between treatment groups were determined by unpaired Student t-tests using Prism 6.2 (Graphpad Software Inc) with significance set at p <0.05. We also tested our data using ANOVA followed by Tukey's posthoc test; this approach yielded the same outcomes.

As illustrated in Figure 1, following maternal exposure to paracetamol (either acute or chronic), there was a large number of genes that significantly altered their expression in the E19 placentas in two-way comparisons to control tissue, with much fewer that changed between the two treatment groups (chronic/acute). Most genes were uniquely regulated, either up or down, depending on treatment duration, with relatively few that were common to both treatment regimes (64 up-regulated and 57 down-regulated). In contrast, in a three-way comparison, only one gene, Nfkbia (NF-kappa-B inhibitor alpha), was shared in all three comparisons ( Figure 1). NFKB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines.

The expression of 121 transcripts (the sum of up-regulated and down-regulated genes in the chronic/acute comparison) was significantly different between acute and chronic treatment groups, suggesting an effect of treatment duration. Of these genes, 34 were significantly up-regulated in chronically treated animals when compared to either the acute treatment group or the control group and eight were down-regulated ( Figure 1).

Comparing datasets of placentas from chronically treated dams with untreated control dams, the expression of 737 genes was significantly different (either up or down p<0.05, see Methods) ( Figure 2). The top 50 up-regulated and down-regulated genes in E19 placentas are displayed for both acute and chronic treatment groups compared to controls in Table 1. In the E19 placentas, many of the top genes up-regulated following chronic treatment were related to immune-response and inflammation ( Table 1, Table 2 and Table 5). It is difficult to determine the extent to which a statistically difference in gene expression is also functionally significant. It is perhaps worth noting that fewer genes were up-regulated two-fold or more with either acute or chronic paracetamol treatment (25 and 34 genes, respectively) compared to the number that were down-regulated two-fold or more (58 and 61, respectively). In addition, the degree of down-regulation was appreciably greater for many of these genes compared with those that were up-regulated. This was particularly evident for the chronically treated group compared to the control group, with five genes down-regulated greater than 500-fold ( Afp, apoc2, rbp4, apob and fgb, Table 1). In addition, 10/50 genes were up-regulated following both treatments but 34/50 were down-regulated following both treatments. Thus overall the down-regulatory effects of paracetamol were much more pronounced than the up-regulatory effects.

Genes that showed a regulatory response in placentas of animals following both acute and chronic treatment with paracetamol are listed in Table 5. Seven of these down-regulated genes showed a fold change of more than two, which was greater in the chronically treated placentas. Other changes were so small that they are unlikely to be of much functional significance.

In the placenta of chronically treated rats there was a notable up-regulation of immune response related genes compared to the acutely treated group ( Table 2). Figure 3 illustrates an analysis from biological Gene Ontology (GO) categories of immune response genes (A) subdivided into the innate (B) and adaptive (C) immune systems in the chronically treated animals. In the placenta these included GO biological processes such as neutrophil chemotaxis (p=4.7E-05) and innate immune response (p=0.045). Figure 3 illustrates that the number of significantly up-regulated genes was substantially more than the number of down-regulated genes and that most of these were in the innate immune system category, with a small number in the adaptive immune system. A list of inflammatory and immune-related genes that were up-regulated in the placenta following chronic treatment is shown in Table 2. Overall, some 36 genes showed a statistically significant up-regulation. These included 15 genes that were up-regulated two-fold or more. As can be seen from Table 2, the third most up-regulated gene in the placenta following chronic paracetamol exposure was Il1ß. Figure 4 illustrates the number of Il1ß gene transcripts in the three treatment groups in the fetal brain and placenta. There was a prominent increase in Il1ß transcripts in the placentas from the dams treated with chronic paracetamol and no difference between the datasets of placentas from the control and acutely treated mothers, both showing very low numbers. IL1ß is a prototypical marker for inflammation and immune response, with up-regulation in the chronically treated placenta of 13.3 fold change; it could thus be a potential indicator of fetal harm. The response in the placenta following a single acute dose of paracetamol was much more muted ( Table 2).

Transcripts per million in E19 fetal placenta and brain from control (n=4), acute (n=4) or chronically treated dams (n=4) as determined by RNA-Seq. (HTSeq-counts, EdgeR). Means ± SD. * p <0.05.

Amongst the down-regulated genes in the placenta ( Table 1) were several transcripts for plasma proteins (AFP, transthyretin and transferrin, see Discussion) that have been shown to down-regulate under inflammatory conditions (negative acute phase response, Heinrich et al., 1990; Hu et al., 2019; Mackiewicz et al., 1990). Two of these were markedly down-regulated in the acute experiments and further down-regulated in the chronic experiments ( Table 1). This suggest that the response of these plasma protein genes was rapid in onset and continuing over several days in the presence of chronic treatment. In contrast, the up-regulatory response of cytokine genes only became prominent in the placentas of animals chronically exposed to paracetamol ( Table 1; Figure 4).

IL1ß concentration (ELISA). In order to see if the increase in transcript numbers for IL1ß in placentas from dams treated chronically with paracetamol ( Figure 4) translated into an increase in its protein concentration, the levels of this cytokine in plasma of both the dams and pups were measured using a commercially available ELISA kit (see Methods). Results are illustrated in Figure 5. None of the dams in any of the treatment groups had a detectable level of IL1ß in their plasma (limit <5pg/ml) nor was IL1ß detected in the control untreated fetuses. In contrast, IL1ß in the plasma of many of the E19 fetuses whose mothers had been treated with paracetamol was detected. The levels were generally higher in fetuses of mothers treated chronically (acute 2/4, chronic low 7/16 and chronic high 10/19).

Samples from control (4 fetuses from 2 dams), acute (4 fetuses from 4 dams) or chronic paracetamol treated dams at low dose (3.75mg/kg, 16 fetuses from 5 dams) or high dose (15mg/kg, 19 fetuses from 7 dams). Measured by ELISA (R&D Quantikine). Means ± SD.

Transcriptomic analysis of the E19 fetal brain was carried out in material collected from the same animals as was prepared for placental analysis, thus allowing a direct comparison between the response of the two tissues to paracetamol treatment of the mother.

As illustrated in Figure 1, following maternal exposure to paracetamol, there was a large number of genes that significantly altered their expression in the fetal brain.

As shown in Figure 2, comparing the dataset for fetal brains from chronically treated dams with untreated control dams, there was a total 1128 genes with significantly different transcript numbers in the E19 brain. The top 50 up-regulated and down-regulated genes in the E19 brain are shown for both acute and chronic treatment groups compared to controls in Table 3. Following both treatments 26/50 genes were up-regulated and 40/50 were down-regulated. Additionally, the level of down-regulation was greater for most transcripts than up-regulation following both acute and chronic paracetamol treatment, for example Col1a1 (collagen type 1 alpha 1 chain) and Col3a1 (collagen type 3 alpha 1 chain), see Table 3. There will be a further analysis of the brain data in a later publication ( Koehn et al., unpublished reports ) that will deal with expression of ABC efflux transporters and related enzymes as these may play a role in the extent to which paracetamol enters the brain at different stages of development ( Koehn et al., 2019b).

In addition to effects of the length of exposure to the drug on gene expression in individual tissues, the regulation in brain and placenta was very different following the same treatment.

Only 98 genes were significantly regulated in both tissues, equating to 5.5% of the transcripts that changed their expression ( Figure 2).

In the E19 placenta many of the top genes up-regulated following chronic treatment were related to immune-response and inflammation, including Il1ß, which was 3 ^rd highest ( Table 1). In contrast, in the brain, very few transcripts for Il1ß ( Figure 4) or other cytokines ( Table 4) could be detected and there was no difference in transcripts for Il1ß between the treatment groups ( Figure 4). Table 4 lists the inflammatory and immune-related genes that were up- or down-regulated significantly in the E19 brain. The changes were very small compared to the placenta in both the innate immune and the adaptive immune category ( Figure 3). Table 5 shows immune/inflammatory related genes that showed a regulatory change in both the placenta and the brains from both acutely and chronically treated fetuses.

No changes in plasma protein transcript numbers were detected in the fetal brain (see Discussion). This, together with lack of up-regulation of the inflammatory cytokine Il1ß, as seen in the placentas, indicates that an inflammatory response was elicited by paracetamol in the placenta but little or none in the fetal brain. We do not have information if other organs not investigated in this study, such as the liver, could also have been affected.

In order to investigate if a prolonged exposure to paracetamol and resulting inflammatory response could affect the permeability of the placenta, two sets of permeability experiments were conducted using a small molecular size marker, ¹⁴C-sucrose (see Methods). These were designed to examine the transfer from the mother to the fetus but also from the fetus back to the mother. Results are illustrated in Figure 6 and Figure 7.

Fetal to maternal transfer of ¹⁴C-sucrose. To investigate the placental transfer of sucrose from fetus back to the dam following maternal paracetamol exposure, sucrose was injected directly into the pups still within their amniotic sacs (see Methods). Two litters were injected in mothers that had been treated with chronic high doses of paracetamol and one litter from a mother treated with chronic low dose paracetamol. These were compared with one litter from an untreated control mother. Plasma samples from both the fetuses and dam were collected and ratio of ¹⁴C- sucrose estimated (see Methods). The results are shown in Figure 6. All three of the litters from mothers treated with chronic paracetamol (either high or low dose) showed slightly higher permeability from the fetus back to the mother than in the control dam. However, the ratios are extremely low, making accurate comparison difficult.

Maternal to fetal transfer of ¹⁴C-sucrose. In order to investigate if the rate of transfer of a small molecular marker from dam to fetus across the placental barrier was affected following paracetamol exposure, dams either untreated (control) or treated with chronic high (15mg/kg) doses of paracetamol were given a final intravenous (i.v.) injection of ¹⁴C-sucrose 30 minutes before removing their fetuses ( Figure 7). Blood samples from dams were time matched to the removal and blood collection from each fetus (see Koehn et al., 2019b). The transfer from the mother to the fetus in the paracetamol treated dams was slightly less than that in the control animal. The much higher ratios obtained in the maternal to fetal transfer experiments ( Figure 7) compared to the fetal to maternal transfer ( Figure 6) are due to the differences in volume of distribution, hence dilution of the marker, when sucrose is injected into the mother or into the fetuses.

Detection of AFP in fetal and maternal plasma. In order to investigate if exposure to paracetamol can also influence the transfer of a protein from the fetal circulation into the maternal blood across the placenta, western blot analysis was made of fetal and maternal plasma samples using cross-reacting antibodies specific for AFP (see Methods). Figure 8A shows the blot that contained both the fetal and maternal samples together with one negative control (non-pregnant female rat). Densitometry measurements are illustrated in Figure 8B together with maternal/fetal ratios. There was no detectable band in the non-pregnant control sample and all maternal samples showed a much lower level of the protein than fetal samples. The levels of the protein in fetal samples did not appear to change between the control and any of the treatment groups ( Figure 8B), but in maternal samples, AFP levels were higher in all chronically treated dams compared to un-treated controls. This was reflected in the ratios of AFP in maternal to fetal plasma ( Figure 8B, right panel) in which all of the chronically treated animals had ratios that were above those in untreated controls and in one acutely treated animal. Prolonged exposure to the drug increased AFP transfer from fetus to dam by about three times compared to the control animals.

Two different molecular size markers ( ¹⁴C-sucrose and plasma proteins) were used to assess any changes in blood-brain barrier permeability following chronic paracetamol treatment of the dam. The samples were obtained from the same experiments as the placental permeability studies.

Transfer of ¹⁴C-sucrose into the brain and CSF following different paracetamol treatment regimes. To investigate the transfer of ¹⁴C-sucrose into the fetal brain after maternal paracetamol exposure, fetal blood, brain and CSF samples from dams untreated (control) or treated acutely or chronically with either low (3.75mg/kg) or high (15mg/kg) doses of paracetamol were measured. As shown in Figure 9, there was no significant difference in the transfer into the brain and CSF between any treatment groups ( Figure 9).

Fetuses were exposed to ¹⁴C-sucrose either directly (fetal i.p. injection) or indirectly (maternal i.v. injection). Treatment groups investigated were control, no paracetamol (n=13), chronic low dose (3.75mg/kg, n=11) and chronic high dose (15mg/kg, n=11) in fetuses that were injected directly. In experiments in which the ¹⁴C-sucrose was injected into the mothers; n numbers were control (n=8), acute (n=10) and chronic high dose (n=6) in the mothers. Means ± SD.

Entry into the brain and CSF when the ¹⁴C-sucrose was injected directly into the fetus was also investigated and results are illustrated in Figure 9. Here too there were no significant differences in the entry of sucrose into brain and CSF between the three treatments.

However, the fetuses that were directly exposed to sucrose (i.p injection) showed a lower level of transfer into the brain and CSF compared to those that were exposed indirectly (i.v. injection to dam), around 10% compared to 40%, respectively. This reflects differences in distribution volume following the different routes of injection as well as the time involved in samples collection.

Blood brain barrier integrity for endogenous plasma protein. Transfer of large molecule plasma proteins into the fetal brain following maternal paracetamol exposure was studied using immunohistochemistry and antibodies to rat serum proteins (see Methods). Brains were matched with plasma samples containing detectable IL1ß levels as estimated by ELISA ( Figure 5). The distribution of the proteins in E19 brains from control, acute and chronic high dose (15mg/kg) paracetamol treated dams is illustrated in micrographs in Figure 10. There was no evidence of a “leak” of protein in any of the vessels in the fetal brains examined. In all sections stained from all brains investigated, immunostaining was exclusively localised in the blood vessels, choroid plexus stroma and precipitated CSF and there was no visible difference in the brain morphology between treatment groups.

Thus, the results clearly show that the blood-brain barrier, at least to plasma proteins and to sucrose, was not affected by paracetamol exposure of the dam, the inflammatory response in the placenta nor the increased levels on IL1ß in fetal blood.

The study has been carried out in pregnant rats at a single gestational age (E19). This stage of brain development in rats at E19 is approximately equivalent to 22–24 weeks gestation in humans ( Clancy et al., 2001), corresponding to the earliest age of viability ( Fischer et al., 2009; Stoll et al., 2010). The rat and human placentas are both classed as hemochorial ( Blood et al., 2007; Dawe et al., 2007) but there are differences in morphology, in particular that the rat placenta has more morphological layers between the fetal and maternal circulations. However, that might mean that the relatively small changes in placental permeability from fetus to mother shown here might be more prominent in the human. The responses of these two species to an inflammatory event are similar with respect to the three plasma proteins AFP, transferrin and transthyretin (prealbumin); as in this study, these proteins have been reported to be acute phase negative proteins under inflammatory conditions ( Heinrich et al., 1990; Hu et al., 2019; Mackiewicz et al., 1990). This supports the suggestion that these findings should be taken account of when advising pregnant women about the use of paracetamol. Given the unexpected findings of up-regulation of inflammatory cytokines and down-regulation of some acute phase plasma proteins, we are in the process of carrying out RNA-Seq replication studies and extending the range of cytokines estimated in fetal and maternal blood. Unfortunately, these experiments have been delayed by the COVID-19 emergency, which has closed our laboratories for an indefinite period. In view of the potential significance of our findings for the use of paracetamol in pregnancy, particularly the high frequency of its use, we feel it is justified to present these findings for peer review, in their present form.

Paracetamol (acetaminophen) is generally considered “safe” to use in pregnancy and lactation ( Australian Medicines Handbook, 2019; Briggs et al., 2017) although it is one of the most commonly overdosed drugs, including in pregnancy (Rayburn et al., 1984). However, some authors urge caution in its use because of evidence of adverse effects ( Brune et al., 2015). It has been reported that as many as nearly 80% of pregnant women in some populations ingest paracetamol ( Dreyer et al., 2015). The findings of the present study, although based solely on experiments in rats, should be taken account of when advising pregnant patients on the use of paracetamol in pregnancy. The clinical situation is not straightforward because in addition to taking paracetamol to relieve pain, it may also be taken to reduce an increase in body temperature accompanying an infection (often respiratory), but there is evidence of an association between infection/fever and adverse outcomes for pregnancies; this seems to be a particular problem when infection/fever occurs at the beginning of the 3 ^rd trimester ( Hagberg et al., 2015). Thus, continued but limited use of paracetamol to control severe pain and to reduce body temperature at critical stages of pregnancy would seems to be appropriate but not the widespread use for lesser indications that is implied by the reports that most pregnant women take paracetamol.

Increased transfer of sucrose and AFP from fetal circulation into maternal circulation, as demonstrated in the present study, suggests that other molecules/metabolites could potentially also reach the maternal circulation. There are several clinical implications, including that increased AFP levels detected in pregnant women are used to detect potential neural tube closure defects, although this test is done earlier in pregnancy and we have as yet no evidence of paracetamol affecting placental permeability this early in pregnancy.

Further investigation is required to see if there are similar effects in the placentas of patients who have taken paracetamol. If the effect is indeed confined to the fetal side of the placenta it will be clinically difficult to determine such an effect in pregnant patients, particularly if it turns out to be variable as in our rat experiments, although transfer of AFP from fetal to maternal circulation might be a useful indicator.

RNA-Seq data on NCBI, Accession number PRJNA633629: https://identifiers.org/ncbi/bioproject:PRJNA633629

Figshare: Effects of paracetamol on rat placenta and fatal brain. https://doi.org/10.26188/5ebff4c2781a0 ( Koehn et al., 2020)

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).