Five-fluorouracil (5-FU) a traditional chemotherapeutic agent used in concurrent chemoradiation therapy (CCRT) to enhance the radiotherapy (RT) effects in rectal cancer patients (Fisher et al., 1988). Compared to surgery or RT alone, adjuvant CCRT (Wolmark et al., 2000) or neoadjuvant CCRT (Bosset et al., 2006) improves the locoregional control and overall survival in rectal cancer patients by 10%–15%.

More than half of the catabolic activity of 5-FU in the liver through the dihydropyrimidine dehydrogenase (DPD) pathway generates toxic 5-fluoro-5,6-dihydro-uracil (5-FDHU), followed by fluoroureidopropionic acid and α-fluoro-β-alanine (Wasternack, 1980; Heggie et al., 1987; Bocci et al., 2000). The cytosolic enzyme DPD in the catabolism of 5-FU is widely expressed in the body (Lu et al., 1993; Van Kuilenburg et al., 1997; Mcleod et al., 1998). The increased AUC ratio of 5-FU/5-FDHU is associated with adverse events (Di Paolo et al., 2001), and the modulation of the catabolic pathway of 5-FU has an impact on the side effects and adverse reactions (Ezzeldin and Diasio, 2004).

Pharmacokinetics is the study of the kinetics of a drug and/or its metabolites in the body and what the body does to the drugs (1991). In the past, RT has been used as a local treatment (Elsasser and Scholz, 2007). Growing evidence shows that the systemic pharmacokinetics (PK) of anticancer drugs can be modulated by local RT with different RT doses; this is called the RT-PK phenomenon (Hsieh et al., 2010; Hsieh et al., 2013; Hsieh et al., 2015; Chen et al., 2017). The area under the plasma concentration versus time curve (AUC) of 5-FU is reduced by RT (Hsieh et al., 2015), and the excretion of 5-FU is facilitated by RT (Hsieh et al., 2011).

However, whether RT modulates the PK of the metabolite of 5-FU, 5-FDHU, remains unclear. The current study investigates the interaction between RT and the pharmacokinetics of 5-FU and its metabolite in rats. The goal of the current study is to provide clinicians with more information about the interaction between RT, 5-FU, and 5-FDHU and to improve daily practice.

The protocol was reviewed and approved by the Institutional Animal Experimentation Committee of National Yang-Ming University, Taipei, Taiwan, and by the Institutional Animal Care and Use Committee (IACUC, approval number 106DN22). Male Sprague-Dawley rats (250–280 g) were provided by the Laboratory Animal Center at National Yang-Ming University (Taipei, Taiwan). The animals had access to water ad libitum and food (laboratory rodent diet 5P14, PMI Feeds, Richmond, IN, USA) and lived in a pathogen-free environment with a 12-h light-dark cycle. All animal experiments followed the guidelines and procedures for the care of laboratory animals at National Yang-Ming University.

The rats were anesthetized with urethane 1 g/ml and achloralose 0.1 g/ml (1 ml/kg by intraperitoneal injection) and were immobilized on a board when undergoing computed tomography for the imaging of the whole pelvic field. Conventional radiotherapy was used to deliver the radiation dose via the anterior-posterior (AP) and PA portals ( Figure 1A ) (Hsieh et al., 2011). The experimental animals were randomized to the control, 0.5 Gy followed by 5-FU and 2 Gy followed by 5-FU groups. Each group’s data were collected from six rats. Dosages of 0.5 and 2 Gy for the rats and 100 mg/kg as a feasible 5-FU dose in rats to examine the 5-FU pharmacokinetic parameters were determined from the results of previous reports (Hsieh et al., 2010; Hsieh et al., 2011; Hsieh et al., 2015). Twenty hours after RT, the rats were administered 100 mg/kg of 5-FU in 2 ml of normal saline by intravenous infusion into the femoral vein over a 2-min period (Hsieh et al., 2010) ( Figure 1B ). A 150-μl blood sample was withdrawn from the jugular vein with a fraction collector according to a programmed schedule at 5, 15, 30, 45, and 60 min and 1.5, 2, 2.5, and 3 h following drug administration. The total blood volume of rodents is approximately 7% of their body weight, and approximately 20% of total blood volume can be withdrawn (Lee and Goosens, 2015). Thus, the volume of a blood sample that can be collected from a Sprague-Dawley rat is approximately 19.6 ml. In the current study, the volume of blood sample for each time collection was 150 μl, so the total blood volume collection was below 10% of the total circulating blood volume to avoid affecting the physiological index of the rats. The blood samples were immediately centrifuged at 3300× g for 10 min. The resulting plasma (50 μl) was added to 1 ml of ethyl acetate in a clean tube, vortexed for 5 min, and centrifuged at 5900 × g for 10 min. After centrifugation, the upper organic layer containing the ethyl acetate was transferred to a new tube and evaporated to dryness under flowing nitrogen. The dried residue was reconstituted with 50 ml of Milli-Q water (Millipore). A 20-µl aliquot of the solution was injected into the high-performance liquid chromatography-ultraviolet (HPLC-UV) detection system.

To collect the bile for a prolonged period, the bile was collected by a duct-cannulated rat model under anesthesia. Exteriorization of a PE10 tube was prepared before bile duct catheterization. A PE10 cannula was fixed to the bile duct (two positions at near liver and duodenum) by a surgical knot, and the wound was closed by sutures after duct cannulation was completed. The bile of the rat flowed into the circulation system when the pipes of the bile ducts were connected. 5-FU was administered via a PE50 cannula into the femoral vein, and blood was collected through a PE50 cannula from the jugular vein.

The pharmacokinetic parameters were determined by calculating each individual set of data with a non-compartmental model using WinNonlin Standard Edition Version 1.1 software (Scientific Consulting Inc., Apex, NC). The pharmacokinetic parameters calculated were the initial drug concentration of 5-FU (C ₀), the maximum concentration and time of 5- FDHU (C _max, t_max), the area under the concentration versus time curve (AUC), the clearance (CL), the elimination half-life (t _1/2), the volume of distribution at steady state (Vss) and the mean residence time (MRT). The statistical analyses were performed using analysis of variance in the SPSS 18.0 program (SPSS Inc., Chicago, USA) and SigmaPlot 10.0 software. All data are expressed as the mean ± standard deviation (S.D.). One-way ANOVA was used for the comparison between groups, and statistically significant differences were defined as ^* P < 0.05 or ^** P < 0.01.

5-FU is commonly used to enhance RT effects (Wolmark et al., 2000; Bosset et al., 2006). The RT-PK phenomenon of 5-FU is a phenomenon in which systemic 5-FU could be modulated by local irradiation with a change in the AUC of 5-FU in plasma (Hsieh et al., 2011; Hsieh et al., 2015). However, whether the metabolism of 5-FU is modulated by RT is still unclear (Hsieh et al., 2015). Here, local pelvic irradiation reduced the AUC_plasma values of 5-FU by 24% and 35% at 0.5 Gy and 2 Gy, respectively. The AUC_plasma values of 5-FDHU, the metabolite of 5-FU, also declined by 21% at 0.5 Gy and 52% at 2 Gy. Additionally, the AUC_bile values of 5-FU increased by 37% and 69% at 0.5 Gy and 2 Gy, respectively. Meanwhile, the AUC_bile values of 5-FDHU increased by 40% at 0.5 Gy and 248% at 2 Gy. The current study reconfirmed the RT-PK phenomenon of 5-FU and suggested that the metabolite of 5-FU, 5FDHU, can be modulated by RT, with similar trends in the two compounds.

There are correlations between plasma levels of 5-FU and treatment outcomes (Milano et al., 1994; Gamelin et al., 1998). Higher AUC values of 5-FU are associated with impressive survival and response rates (Milano et al., 1994; Gamelin et al., 1998). However, 31% to 34% of treated patients have dose-limiting toxicities (Meta-Analysis Group In et al., 1998). Concomitant administration of 5-FU and RT increases the rate of observed grade 3 or higher acute mucositis (Strigari et al., 2016). Bazan et al. (2013) reported that acute hematologic toxicity in patients treated with pelvic RT concomitant with 5-FU was 8% higher than in those undergoing RT alone. Prolonged tumor retention of 5-FU and the enhanced cytotoxicity followed by RT was reported by Blackstock et al. (1996).

Actually, as the ratio of AUC of 5-FU/5-FDHU increased, the risk of adverse events in cancer patients also increased (Di Paolo et al., 2001). The current study showed that the 5-FU/5-FDHU AUC ratios in the sham, 0.5 Gy and 2 Gy group were 13, 13 and 18, respectively. Additionally, pelvic irradiation significantly increased the respective Vss of 5-FU by 16.7% at 0.5 Gy and 17.7% at 2 Gy. According to the above data (Blackstock et al., 1996; Di Paolo et al., 2001), the current RT-PK phenomenon supported that RT plus 5-FU could cause more adverse events than 5-FU alone by increasing the ratio of AUC of 5-FU/5-FDHU and the Vss of 5-FU. Additionally, the off-target dose also modulates the PK of 5-FU and contributes to the toxicity during CCRT.

Bocci et al. (2006) noted that there are significant correlations between the AUCs of 5-FU and 5-FDHU. Additionally, 5-FDHU plasma levels may correlate with DPD activity weakly but significantly (Bocci et al., 2006). Meanwhile, DPD activity is determined by the CL of 5-FU in plasma, and high-5-FU CL predicts lower toxicity and poor outcome for colorectal cancer patients (Gusella et al., 2009). The current study showed that pelvic irradiation, whether 0.5 Gy or 2 Gy, increased the CL of 5-FU. Meanwhile, the AUC_plasma of 5-FDHU declined by both irradiation doses. Additionally, the CL of 5-FDHU also increased by irradiation. In contrast to the values in the plasma, the AUCs of 5-FU and 5-FDHU in the bile were increased by irradiation. This suggests that the activity of DPD may be decreased by irradiation and may be dose-dependent. Irradiation may reduce the toxicity of 5-FU by decreasing the AUC_plasma of 5-FU and increasing the bile excretion of 5-FU. However, the decreased AUC of 5-FU may cause poor clinical outcomes (Milano et al., 1994; Gamelin et al., 1998; Gusella et al., 2009), suggesting that RT followed by 5-FU is not an ideal model in clinical practice.

There were no differences in the levels of TGF-β1 and TNF-α between the RT and sham groups. However, the expression of MMP-8 increased by 100% at 2 Gy when compared with the sham group. Irradiation causes bystander signaling or abscopal effects through interleukins, cytokines, reactive oxygen species, TGF-β1 and TNF-α (Chen et al., 2017). MMPs degrade extracellular matrix proteins. MMP activity can be upregulated by nitric oxide (NO)-mediated S-nitrosylation (Gu et al., 2002). MMP-8 is an proinflammatory mediator and mediates inflammatory processes (Lee et al., 2014). MMP-8 acts directly on the proinflammatory cytokine TNF-α, leading to cytokine production and inflammatory responses (Sriram and O’callaghan, 2007; Bahia and Silakari, 2010); MMP-8 is upregulated during hepatic ischemia and reperfusion injury (Cursio et al., 2002). A previous report suggested that MMP-8 may play a role in the RT-PK phenomenon (Hsieh et al., 2011). Interestingly, the current study confirmed the upregulated expression of MMP-8 in the plasma after local irradiation. However, there is very little known about the specific functions of MMP-8 in the RT–PK phenomenon.

Low-dose RT in clinical practice has become more popular with advanced radiotherapy techniques. However, off-target RT may produce unexpected or unwanted biological effects (Shueng et al., 2009; Darby et al., 2013). For example, the increased scattering of low-dose irradiation by esophageal stent during treatment increases the risk of aortic pseudoaneurysm formation and the risk of perforating the esophagus (Hou et al., 2015; Lu et al., 2018). An additional 1 Gy of radiation increases the incidence of major coronary events by 7.4% in breast cancer patients receiving RT (Darby et al., 2013). However, the biological effects of low-dose RT are still unclear. Here, the current study confirmed that even low-dose irradiation can modulate the PK of anticancer drugs and their metabolites and can facilitate the excretion of drugs to the bile.

There were some limitations to this study. First, the current study was designed to examine the interaction between RT and PK of 5-FU and 5-FDHU but did not include the pharmacodynamics of 5-FU and 5-FDHU during RT. The current study confirmed there were no significant differences of toxicity effects between 5-FU-treated and RT plus 5-FU-treated groups. The current data was compatible with the previous report (Hsieh et al., 2011). These data also provided clues about systemic toxicology during the RT-PK phenomenon. Second, the delivery of RT in the current study was a single fraction. Further study to mimic clinical practice by using continued RT delivery to optimize the timing, duration, and dosing of 5-FU and 5-FDHU in the RT-PK phenomenon is warranted. Third, the possible mechanism was not included in the current study, although the current study noted that MMP-8 expression in the plasma was upregulated in the 5-FU and 5-FDHU following RT group. However, we confirmed that the systemic PK of 5-FU and 5-FDHU could be modulated by RT with sequential administration. Finally, the decreased AUC of 5-FU may cause poor clinical outcomes; nevertheless, it is not supported by the current experimental evidence. The current results just confirm that irradiation could modulate the pharmacokinetics of 5-FU and 5-FDHU. Further studies for detecting the optimal strategies, such as metronomic (Bocci and Kerbel, 2016), concurrent or sequential regimens for RT and 5-FU, are clearly still required in the future.

The current results show that both conventional and low-dose irradiation can modulate the pharmacokinetics of 5-FU and 5-FDHU. Additionally, RT plus 5-FU could cause more adverse events than 5-FU alone by increasing the AUC ratio of 5-FU/5-FDHU and the Vss of 5-FU. Irradiation may reduce the toxicity of 5-FU by decreasing the AUC of 5-FU in the plasma and increasing the bile excretion of 5-FU. However, the decreased AUC of 5-FU may cause poor clinical outcomes, suggesting that a sequential regimen of RT and 5-FU is not an ideal model in clinical practice. Hopefully, the current study sheds new light on these effects and increases our understanding of the effects of low dosage in the era of highly advanced RT.

All datasets generated for this study are included in the article.

The animal study was reviewed and approved by The Institutional Animal Experimentation Committee of National Yang-Ming University, Taipei, Taiwan, and by the Institutional Animal Care and Use Committee (IACUC, approval number 106DN22).

All of the authors have read and approved the final manuscript. Study design and data interpretation: J-HL, T-HT, and Y-JC. Data collection: J-HL. Data analysis: J-HL. Manuscript writing: J-HL and C-HH. Manuscript revision: L-YW and H-YL. Acquisition of funding: T-HT, Y-JC, L-YW, and C-HH.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.