Comparative NMR-Based Metabonomic Investigation of the Metabolic Phenotype Associated with Tienilic Acid and Tienilic Acid Isomer

An NMR-based metabonomic approach was applied to study the systems level metabolic effects of two closely related thiophene compounds, tienilic acid (TA) and tienilic acid isomer (TAI). The metabonomic data were anchored with traditional clinical chemistry and histopathologic analyses. TA was removed from the market as a result of suspected immune-mediated hepatotoxicity, whereas TAI is an intrinsic hepatotoxin. Equimolar doses of TA and TAI were administered to Sprague–Dawley rats, and sampling was conducted at 2, 6, and 24 h post-treatment. Histopathologic analyses revealed development of a significant hepatic lesion 24 h post-TAI treatment with a parallel increase in plasma alanine aminotransferase (ALT) activity. In contrast, TA was not associated with the development of a hepatic lesion or an increase in plasma ALT activity. High-resolution NMR spectral metabolic profiles were generated for liver extracts, plasma, and urine at multiple time points. Multivariate statistical tools were applied to model the metabolic profiles and identify discriminatory metabolites that reflected both the adaptation to TA administration and the onset and progression of TAI-induced hepatotoxicity. TAI was shown to induce marked metabolic effects on the metabolome at all time points, with dramatic metabolic perturbations at 24 h post-treatment correlating with the histopathologic and clinical chemistry evidence of a hepatic lesion. The TAI-induced metabolic perturbations provided evidence for the generation of electrophilic reactive metabolites and a significant impairment of bioenergetic metabolic pathways. TA induced early metabolic perturbations that were largely resolved by 24 h post-treatment, suggesting the reestablishment of metabolic homeostasis and the ability to adapt to the intervention, with hepatic hypotaurine potentially representing a means of assessment of hepatic adaptation. This comparative metabonomic approach enabled the discrimination of metabolic perturbations that were common to both treatments and were interpreted as nontoxic thiophene-induced perturbations. Importantly, this approach enabled the identification of temporal metabolic perturbations that were unique to TAI or TA treatment and hence were of relevance to the development of toxicity or the ability to adapt. This approach is applicable to the future study of pharmacologically and structurally similar compounds and represents a refined means of identification of biomarkers of toxicity

Protein absorbance (A₂₈₀) and HCTLase activity of column fractions from the chromatographic purification of human liver HCTLase (A–E) and recombinant HCTLase (F, G).

<p>A₂₈₀ nm, solid lines; HCTL activity, solid lines with open circles. (A) Carboxymethyl (CM) column 1, (B) ceramic hydroxyapatite (HA) column, (C) Superdex 200 column 1, (D) CM column 2, (E) Superdex 200 column 2; (F) HA column, (G) Superdex 200 column. Ticks represent fraction changes.</p

Inhibition of human liver HCTL activity.

<p>* HCTLase activity was measured at 10 mM concentration of substrate.</p><p>† Iodoacetamide inhibited rBPHL to the same extent.</p><p>Inhibition of human liver HCTL activity.</p

IEF activity and Coomassie stain of liver BPHL and rBPHL.

<p>(A) HCTLase activity stain of rBHPL (lane 2) and human liver BPHL (lane 3). Hemoglobin shows a faint band as well (lane 1). (B) Coomassie Blue stain of hemoglobin, rBPHL and human liver BPHL (lanes 1–3, respectively).</p

LC-MS chromatograms showing rBPHL-mediated HCTL cleavage at (A) t = 5 min and (B) t = 0 of reaction time, and valacyclovir ester cleavage at (C) t = 5 min and (D) t = 0.

<p>Chromatograms A and B represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 117 > 89 (for HCTL, Rt  =  10.7 min) and <i>m/z</i> 136 > 89 (for Hcy, Rt  =  5.4 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 136) of the product peak at Rt  =  5.4 min, and is consistent with the spectrum obtained from commercial Hcy. Chromatograms C and D also represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 326 > 152 (for VC, Rt  =  18.3 min) and <i>m/z</i> 226 > 152 (for acyclovir, Rt  =  6.1 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 226) of the product peak at Rt  =  6.1 min, and is consistent with the spectrum obtained from commercial acyclovir. The small product peak at t  =  0 in chromatogram D reflects the extremely rapid metabolism of VC by rBPHL, which generated a detectable amount of acyclovir in even the very short period of time (∼10 sec) between initiation and quenching of the enzymatic reaction.</p

SDS-PAGE analysis of pooled column fractions from (A) human liver and (B) recombinant HCTLase purification.

<p>(A) Lane 1, molecular weight markers (kDa); lane 2, liver extract; lane 3, diethylaminoethyl (DEAE) pool; lane 4, CM-1 pool; lane 5, ceramic hydroxyapatite (HA) pool; lane 6, Superdex 200 (#1) pool; lane 7, CM-2 pool; lane 8, Superdex 200 (#2) pool. Gel was stained with silver stain kit (Pierce Chemical). (B) Lane 1, molecular weight markers (kDa); lane 2, <i>E. coli</i> extract; lane 3, DEAE pool; lane 4, HA pool; lane 5, Superdex 200 pool. Gel was stained with Imperial Protein Stain (Pierce Chemical).</p

Purification of recombinant human BPHL (HCTLase).

<p>DEAE indicates diethylaminoethyl; and HA, hydroxyapatite.</p><p>* Units of HCTLase activity are µmol NTB produced per minute measured at ≈K_m concentration of substrate.</p><p>Purification of recombinant human BPHL (HCTLase).</p

BPHL substrates.

<p>* <i>V</i>_max from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone-0110054-g004" target="_blank">Figure 4B</a>.</p><p>† from this study.</p><p>‡ from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone.0110054-Puente2" target="_blank">[27]</a>.</p><p>BPHL substrates.</p

Substrate dependence and pH optimum of HCTL hydrolysis by BPHL.

<p>Substrate dependence of HCTL hydrolysis by (A) purified human liver HCTLase (BPHL) and (B) rBPHL. Closed symbols, velocity (v) vs substrate concentration [S]; open symbols, [S]/v vs [S]. K_m for HCTL hydrolysis by purified liver BPHL =  3.92 mM and for rBPHL K_m  =  3.18 mM. (C) HCTLase activity of rBPHL (% maximal activity) as a function of pH. Open squares, □―□ 50 mM citrate/Na₂HPO₄; open circles ○―○, 50 mM Tris-HCl; open triangle, ▵―▵ 50 mM Na₂PO₄.</p