Table E.1.
In vitro studies on biotransformation of OTA
| Experimental system | Species | Purity of OTA | Metabolites | Analytical method | Structure confirmation | Comment | Reference |
|---|---|---|---|---|---|---|---|
| Liver microsomes | Rabbit, human, pig | 4(R)‐HO‐OTA;4(S)‐HO‐OTA | TLC | MS, NMR | Enzyme kinetics (Vmax + KM) | Størmer et al. (1981) | |
| Liver microsomes +/− phenobarbital | Rabbit | Not specified | 4(R)‐HO‐OTA; 4(S)‐HO‐OTA; 10‐HO‐OTA |
TLC HPLC‐UV |
MS, NMR | Enzyme kinetics (Vmax + KM) | Størmer et al. (1983) |
| Kidney cells (Vero cells) | Monkey | Not specified | OTalpha; 4(R)‐HO‐OTA; 4(S)‐HO‐OTA; OTB | HPLC‐FLD | – | No kinetics | Grosse et al. (1995) |
| Kidney microsomes +/− phenobarbital | Rabbit | Not specified |
4(R)‐HO‐OTA; 4(S)‐HO‐OTA 10‐HO‐OTA; OTB; 3 unidentified metabolites |
HPCL‐FLD | No kinetics | El Adlouni et al. (2000) | |
| Bronchial epithelial cells +/− phenobarbital | Human | Not specified | OTalpha; 4(R)‐HO‐OTA; 4(S)‐HO‐OTA; 10‐HO‐OTA; OTB; 3 unidentified metabolites | ||||
| Liver microsomes +/− GSH and cytosol | Rat ♂♀ | [U‐3H]OTA (18 Ci/mmol) radiochemical purity > 98% | 4(R)‐HO‐OTA | HPLC‐radioactivity detector | MS, NMR | Enzyme kinetics (Vmax) | Gautier et al. (2001) |
| Liver microsomes +/− GSH and cytosol | Human | 4(R)‐HO‐OTA | No evidence of GSH conjugates | ||||
| Liver S‐9 +/− GSH | Rat | 4(R)‐HO‐OTA | No evidence of GSH conjugates | ||||
| Kidney microsomes +/− GSH and cytosol | Rat, Human | – | No evidence of GSH conjugates | ||||
| CYP1A1, 1A2, 2E1, 3A4 | Human | 4(R)‐HO‐OTA | |||||
| CYP1A2, 2C11 | Rat | – | |||||
| Seminal vesicle microsomes | Ram | Unidentified metabolite | |||||
| Seminal vesicle microsomes enriched in prostaglandin H‐synthase Horseradish peroxidase | – | ||||||
| Liver microsomes +/− inducers of CYPs | Rat, mice ♂♀ | > 99% (HPLC) | 4(R)‐HO‐OTA; 4(S)‐HO‐OTA | HPLC‐FLD | MS/MS | Enzyme kinetics (Vmax + KM) | Zepnik et al. (2001) |
| CYP1A2, 2C9‐1, 3A4 supersomes® a | Human | > 99% (HPLC) | 4(R)‐HO‐OTA; 4(S)‐HO‐OTA | MS/MS | Enzyme kinetics (Vmax) | ||
| Semipurified GSTs +/− liver and kidney cytosol | Rat | > 99% (HPLC) | 4(R)‐HO‐OTA; 4(S)‐HO‐OTA | MS/MS | Enzyme kinetics (Vmax) | ||
| Kidney microsomes or cytosol fortified with NADPH and GSH; Liver cytosol | Rat | > 99% (HPLC) | – | – | – | ||
|
Horseradish peroxidase/H2O2 Soybean lipoxygenase |
– | > 99% (HPLC) | – | – | – | ||
| Primary hepatocytes +/− induction by 3‐methylcholanthrene | Rat, human | 3H‐OTA (0.6 Ci/mmol) |
4(R)‐HO‐OTA; OTA‐hexose OTA‐pentose |
HPLC‐radioactivity detection/quantification system | MS/MS | No effect of β‐Gluc, sulfatase or GGT on metabolite retention times, suggesting that phase‐II‐conjugates were not formed | Gross‐Steinmeyer et al. (2002) |
| Liver microsomes | Rat | Not specified | 4‐HO‐OTA; OTHQ‐GSH | LC‐MS/MS | OTHQ‐GSH generated in less than 1% yield | Dai et al. (2002) | |
| Horseradish peroxidase/H2O2 | – | ||||||
| Fe2(NH4)2(SO4)2/H2O2 | OTHQ‐GSH | ||||||
| Kidney cells | Opossum | Not specified |
OTalpha; 4(R)‐HO‐OTA 4(S)‐HO‐OTA; OTB; OTHQ; OP‐OTA; Several unidentified metabolites |
HPLC‐FLD nano‐ESI‐IT‐MS |
No kinetics | Faucet‐Marquis et al. (2006) | |
| Liver microsomes | Rat, human, pig, cow, chicken, goat | 99% |
4(R)‐HO‐OTA; 4(S)‐HO‐OTA Hydroxylated metabolites tentatively identified as 9′‐HO‐OTA: 7′‐HO‐OTA, 5′‐HO‐OTA; OTB |
UPLC‐Q/TOF‐MS |
No enzyme kinetics No OTA‐derived glucuronides detected |
Yang et al. (2015) |
GGT:γ‐glutamyltranspeptidase; β‐Gluc:β‐glucuronidase; GSH: glutathione; HO‐OTA: hydroxy ochratoxin; HPLC‐FLD: high‐performance liquid chromatography with fluorescence detection; KM:Michaelis constant, concentration of substrate which permits the enzyme to achieve half Vmax; NADPH: nicotinamide adeninedinucleotide phosphate; OTA: ochratoxin A; OTalpha: ochratoxin alpha; OTB: ochratoxin B; OTHQ: ochratoxin hydroquinone; LC‐MS/MS: liquid chromatography‐mass spectrometry and tandem mass spectrometry; MS: mass spectroscopy; nano‐ESI‐IT‐MS: TLC: thin layer chromatography; TLC HPLC‐UV: thin layer chromatography high‐performance liquid chromatography with ultraviolet detection; UPLC‐Q/TOF‐MS: ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry; Vmax: maximum velocity of an enzymatic reaction.
Corning Supersomes® enzymes are recombinantly expressed drug metabolising enzyme reagents, consisting of microsomes prepared from insect cells infected with a virus engineered to express a CYP isoform.