Cyclophosphamide (CYP) is one of the most widely used chemotherapeutic agents, and the metabolite acrolein is excreted in urine and accumulates in the bladder to produce toxic effects on the bladder wall, resulting in hemorrhagic cystitis (HC).  HC may be fatal despite treatment options such as hydration, clot extraction via cystoscopy, continuous bladder irrigation, and cystectomy.  Administration of 2-mercaptoethane sodium sulphonate (mesna) and hyperhydration are the two most frequently used methods to prevent HC, but they are not always effective and cannot be used in all patients.  Chemotherapy with CYP is often limited by urotoxicity and requires new drugs against the HC.  Because CYP-induced HC is mediated by an inflammatory process, new compounds with anti-inflammatory properties may be beneficial in reducing side effects during CYP therapy.
5-Aminolevulinic acid (5-ALA) is a natural amino acid that originally exists in the body.  5-ALA is metabolized to protoporphyrin IX (PpIX) and by the insertion of a ferrous ion into PpIX, heme and hemeproteins are produced.  These 5-ALA-derived molecules play important roles in the energy generating function of mitochondria [1], so simultaneous administration of 5-ALA and sodium ferrous citrate (5-ALA/SFC) reportedly improves mitochondrial function by upregulating the expression of hemeproteins involving in respiratory chain complexes and increases the expression level of heme oxygenase-1 (HO-1), a cytoprotective molecule showing anti-oxidative stress and anti-inflammatory effects [2].
Mitochondrial metabolic pathways are important modulators of inflammatory pathways and mitochondrial dysfunction is implicated in aging and pathologies including type 2 diabetes, neurodegeneration, cancer, and inflammatory disorders.  Such cellular senescence is thought to be partly attributable to mitochondrial dysfunction and the associated increase in production of reactive oxygen species, and one of the etiologies of lower urinary tract dysfunction (LUTD) is thought to be increased oxidative stress due to mitochondrial dysfunction in the bladder.  Inflammation and mitochondrial dysfunction may therefore provide therapeutic targets for LUTD.  In this study, therefore, we investigated effects of 5-ALA/SFC pretreatment on bladder dysfunction in CYP-treated rats.

At 13–15 weeks old, male Wistar rats were randomly divided into four groups as follows: (1) Vehicle-1 + Vehicle-2 group (V1+V2); (2) Vehicle-1 + CYP group (V1+CYP); (3) low 5-ALA/SFC + CYP group (ALA100+CYP); and (4) high 5-ALA/SFC + CYP group (ALA300+CYP).  In each rat, Vehicle-1 (saline, 10 ml/kg/day) or 5-ALA/SFC (low: 100 and 157 mg/kg/day; high: 300 and 471 mg/kg/day) was orally administered once daily for 7 days.  On Day 6, Vehicle-2 (saline, 5 ml/kg) or CYP (150 mg/kg) was administered intraperitoneally.
On Day 8 (one day after the last administration of 5-ALA/SFC or Vehicle-1), under urethane anesthesia (0.8 g/kg, ip), cystometry (saline, 4 ml/h) was performed via a catheter inserted into the bladder from the bladder dome.  After cystometry, bladder tissues were collected to perform hematoxylin and eosin staining, ELISA and real-time PCR.

CYP significantly shortened intercontraction intervals (ICI), decreased bladder compliance (Comp), and increased the number of non-voiding contractions (NVCs) (Table 1) without altering post-voided residual volume (Rv) (data not shown).  These CYP-induced changes in ICI and the number of NVCs were significantly attenuated by pretreatment with 5-ALA/SFC at higher doses, and there was no significant difference in Comp between V1+V2 and ALA300+CYP (Table 1).  Therefore, histological and in vitro analyses of bladder tissues were performed in three groups as follows: V1+V2, V1+CYP, and ALA300+CYP.
In histological analysis, CYP increased pathological scores (neutrophil infiltration, bleeding and edema) in the bladder, and pretreatment with higher doses of 5-ALA/SFC significantly improved CYP-induced increases in neutrophil infiltration/bleeding scores (Fig. 1).  In vitro analysis, CYP increased protein levels of myeloperoxidase (MPO), a marker of neutrophil infiltration, in the bladder, however, there was no significant difference in the MPO levels between V1+V2 and ALA300+CYP (Fig. 2).  CYP administration showed no significant change in HO-1 mRNA levels in the bladder, while the HO-1 levels were significantly higher in ALA300+CYP than those in V1+V2 (Fig. 2).

CYP injection significantly shortened ICI, decreased Comp, and increased the number of NVCs without changing Rv, suggesting that CYP successfully induced urinary frequency with low bladder distensibility and detrusor overactivity (DO) in rats.  Pretreatment with 5-ALA/SFC at higher doses significantly attenuated the CYP-induced changes in ICI and the number of NVCs, and the CYP-induced decrease in Comp was not detected in ALA300+CYP.  These data suggest that 5-ALA/SFC pretreatment could improve the urinary frequency, decreased bladder distensibility, and DO in CYP-treated rats.  
In histological and in vitro analyses of rat bladder tissues, CYP significantly increased histological scores of neutrophil infiltration, bleeding and edema, and MPO protein levels, indicating that CYP successfully induced HC.  These CYP-induced changes in scores of neutrophil infiltration and bleeding were significantly attenuated by pretreatment with 5-ALA/SFC at higher doses, and the CYP-induced increase in the MPO levels was not detected in ALA300+CYP.  These data indicate that 5-ALA/SFC pretreatment could partially improve the bladder inflammation induced by CYP.  In addition, CYP administration tended to increase HO-1 mRNA levels, but not significantly, in the bladder, while the HO-1 levels were significantly higher in ALA300+CYP than those in V1+V2.  Because HO-1 is reported to show anti-oxidative stress and anti-inflammatory effects [3], our present data indicate that pretreatment with 5-ALA/SFC improved the bladder inflammation perhaps via the increased HO-1.

Our present data suggest that pretreatment with 5-ALA expects protective effects on bladder dysfunction in CYP-induced HC by partially improving bladder inflammation perhaps via HO-1 up-regulation. Therefore, 5-ALA pretreatment might be useful for prevention of CYP-induced urinary side effects during chemotherapy.

Ota U, Hara T, Nakagawa H et al. 5-Aminolevulinic acid combined with ferrous ion reduces adiposity and improves glucose tolerance in diet-induced obese mice via enhancing mitochondrial function. BMC Pharmacol Toxicol. 2017;18:7
Ito H, Nishio Y, Hara T et al. Oral administration of 5-aminolevulinic acid induces heme oxygenase-1 expression in peripheral blood mononuclear cells of healthy human subjects in combination with ferrous iron. Eur J Pharmacol. 2018;833:25-33.
Fujino M, Nishio Y, Ito H et al. 5-Aminolevulinic acid regulates the inflammatory response and alloimmune reaction. Int Immunopharmacol. 2016;37:71-8.

Continence 2S2 (2022) 100219
DOI: 10.1016/j.cont.2022.100219