Pharmacological inhibition of ALDH1A in mice decreases all-trans retinoic acid concentrations in a tissue specific manner

Abstract

all-trans retinoic acid (atRA), the active metabolite of vitamin A, is an essential signaling molecule. Specifically the concentrations of atRA are spatiotemporally controlled in target tissues such as the liver and the testes. While the enzymes of the aldehyde dehydrogenase 1A family (ALDH1A) are believed to control the synthesis of atRA, a direct relationship between altered ALDH1A activity and tissue atRA concentrations has never been shown. To test whether inhibition of ALDH1A enzymes decreases atRA concentrations in a tissue specific manner, the potent ALDH1A inhibitor WIN 18,446 was used to inhibit ALDH1A activity in mice. The ALDH1A expression, atRA formation kinetics, ALDH1A inhibition by WIN 18,446 and WIN 18,446 disposition were used to predict the time course and extent of inhibition of atRA formation in the testis and liver. The effect of WIN 18,446 on atRA concentrations in testis, liver and serum were measured following single and multiple doses of WIN 18,446. ALDH1A1 and ALDH1A2 were responsible for the majority of atRA formation in the testis while ALDH1A1 and aldehyde oxidase contributed to atRA formation in the liver. Due to the different complement of enzymes contributing to atRA formation in different tissues and different inhibition of ALDH1A1 and ALDH1A2 by WIN 18,446, WIN 18,446 caused only a 50% decrease in liver atRA but testicular atRA decreased over 90%. Serum atRA concentrations were also reduced. These data demonstrate that inhibition of ALDH1A enzymes will decrease atRA concentrations in a tissue specific manner and selective ALDH1A inhibition could be used to alter atRA concentrations in select target tissues.

Keywords: Aldehyde dehydrogenase; Aldehyde oxidase; Liver; Mass spectrometry; Retinoic acid; Testes.

Figure 1. Quantification of ALDH1A expression in mouse liver and testis using LC-MS/MS peptide quantification

Recombinant human ALDH1A protein was digested with trypsin and two peptides generated from each protein were monitored using LC-MS/MS (A,B,C). Representative chromatograms of ALDH1A1 peptide detection in the liver (D) and ALDH1A2 in the testis (E) are shown. ALDH1A3 was not detected in the liver or the testis. Liver and testes from eight mice not treated with WIN 18,446 were used to determine the average ALDH1A isoform expression in each tissue and the measured concentrations are shown with a box and whiskers plot with Tukey distributions in Panel F. ALDH1A1 concentration in the liver was 170 ± 50 pmol/g and in the testis 150 ± 30 pmol/g and the average ALDH1A2 concentration in the testis was 17.0 ± 5.0 pmol/g.

Figure 2. WIN 18,446 inhibits the formation of at RA in mouse liver and testis

The concentration dependent inhibition kinetics of atRA formation in mouse testis (A) and liver (B) S10 protein by WIN 18,446. The NAD⁺ dependent atRA formation was detected in mouse liver cytosol (C) but not in mouse liver microsomes (Panel C inset). The aldehyde oxidase inhibitor hydralazine inhibited 45 ± 2% of the atRA formation while WIN 18,446 inhibited 60 ± 2% of atRA formation. Inhibition of atRA formation in mouse liver cytosol by WIN 18,446 and hydralazine is shown in panel (D). When hydralazine was combined with WIN 18,446, atRA formation was reduced by 95 ± 1% (D). Time dependent inhibition of atRA formation by WIN 18,446 was observed in pooled testis S10 protein, but not liver S10 protein (E). Since time dependent inhibition was observed in the testis S10 protein, the time dependent inhibition of atRA formation by WIN 18,446 was characterized. The rate of inactivation in testis S10 protein was determined with increasing concentrations of inhibitor (F inset) and plotted as a function of inhibitor concentration (F) to the determine the K_I of 420 ± 190 nM and k_inact of 23.2 ± 3.5 hr⁻¹. All experiments were conducted as described in materials and methods.

Figure 3. The disposition of WIN 18,446 in mice following single and multiple doses and predicted effects of WIN 18,446 on ALDH1A1 and ALDH1A2 activity

WIN 18,446 serum concentrations were measured after a single (A) or multiple (B) doses of 125 mg/kg WIN 18,446 given as an oral dose. The equation [WIN 18,446](t)= A^−αt + B^−βt + C^−γt was fitted to the data (dashed line) for the single dose and was used to simulate WIN 18,446 concentrations. The simulated WIN 18,446 concentrations following a single dose of WIN 18,446 were used to predict the time course of ALDH1A1 (C) and ALDH1A2 (D) activity as described in materials and methods.

Figure 4. The predicted effect of WIN 18,446 administration on atRA CL_f and ALDH1A expression in mouse liver and testis

The atRA CL_f was predicted using measured WIN 18,446 disposition data, WIN 18,446 inhibition kinetics of recombinant ALDH1A, and atRA formation in mouse tissues as described in materials and methods. The total liver and testis atRA formation clearances were predicted with a dynamic model after a single dose of WIN 18,446 (A,B) or static model after multiple doses (C,D). The dashed line in A and B represents the total average predicted atRA CL_f in the absence of WIN 18,446 administration. ALDH1A1 expression in the liver and testis and ALDH1A2 expression in the testes in control mice and in mice treated for 8 days with WIN 18,446 are shown (E). The expression levels were not significantly altered by WIN 18,446 treatment (E). The atRA formation velocities were measured in testes S10 fractions from control mice and mice sacrificed 24 hours after a single dose of WIN 18,446, or 24 hours after the 7^th dose of WIN 18,446 (F). The decrease in activity represents the inactivation of ALDH1A activity by WIN 18,446.

Figure 5. at RA concentrations are decreased in a tissue specific manner following single and multiple doses of WIN 18,446

Tissue and serum atRA concentrations were measured using LC-MS/MS over a 24 hour time course after single 125 mg/kg oral dose (A,B,C) and 8 daily doses (B,E,F) of 125 mg/kg WIN 18,446. The dashed lines represent the average concentration of atRA at time 0 of vehicle treated mice. Significant changes in atRA concentrations at any given time point in comparison to those measured at time 0 in the vehicle treated mice are indicated. (p- values: * < 0.05, ** <0.01, *** < 0.001)