Accurate measurement of enzyme activities in porcine liver microsomes is important in e.g. endocrinological and biomedical research. Furthermore, it is essential to carefully validate analytical methods for such measurements prior to routine use. The method used in the present study is an adaptation of recently published methods for rapid determination of EROD and MROD activities in the microsomes from various species [10
]. Here, microsomes from entire and castrated male pigs were used.
3.2. Validation of microsomal incubation
Pooled, rather than individual, microsomal preparations from entire and castrated male pigs were used to select suitable incubation time and microsomal protein content for subsequence analyses. Linearity of resorufin formation from 7-ethoxyresorufin was demonstrated up to 7 min of incubation time and 0.3 mg of microsomal protein. Linearity of resorufin formation from 7-methoxyresorufin was up to at least 20 min of incubation time and 0.6 mg of microsomal protein. Microsomal protein of 0.2 mg and incubation time of 5 min were chosen for the subsequent analysis. Resorufin was not detected in the incubations without NADPH, without substrate or without microsomal protein.
The mean recoveries of resorufin ranged between 77.8 – 107.1% (Table 1
), which indicates that the method is accurate. The lowest recovery of 77.8% was obtained with lowest concentration of resorufin. In the present study, matrix did not affect the measurements. The differences between blank incubations and incubations of resorufin in the presence of microsomes did not exceed 10%.
The linear concentration range of the assay was from 0.5 to 50 pmol/mL (Figure 1
). Inter-assay variations varied from 3.6 to 15.4% (Table 2
). Intra-assay variations did not exceed 12%. The limit of quantitation of resorufin was 0.5 pmol/mL. Taken together, those results demonstrated excellent linearity over the concentration range tested, good accuracy and repeatability, suggesting that the method can be successfully applied to determine EROD and MROD activities in porcine liver microsomes.
Resorufin concentrations in supernatants were stable for at least 5 days if stored at 4 °C under dark conditions. RSD for 8 samples varied from 4.4 to 21.4%; notably the highest RSD (21.4%) at day 5 was obtained when using the sample with the lowest concentration of resorufin (0.9 pmol/ml). At day 8, concentrations of resorufin increased probably due to evaporation of methanol and RSD varied from 7.6 to 26.1%. If stored at room temperature, concentrations of resorufin in the supernatants were stable for at least 1 day (RSD varied from 3.0 to 5.3%).
3.3. Kinetic characteristics of EROD and MROD activities
Eadie-Hofstee plots of EROD activities in porcine liver microsomes revealed a biphasic response, indicating that multiple enzymes are responsible for the biotransformation of 7-ethoxyresorufin to resorufin (Figure 2
). It has been shown in various studies that EROD activities can be related to both CYP1A1 and CYP1A2 isoenzymes. Messina et al
] demonstrated that CYP1A2 purified from β-naphthoflavone treated pigs was catalytically active toward 7-ethoxyresorufin and 7-methoxyresorufin. In rat, mouse and penguin liver microsomes, EROD activities followed a biphasic kinetic pattern [10
]. In contrary, a single enzyme was responsible for EROD in human, monkey and bovine liver microsomes [10
As shown in Table 3
values for the high-affinity components were fairly constant in entire and castrated male pigs. However, Km
for the low-affinity component of EROD and for MROD were somewhat lower in entire male pigs. The reason for this is unclear; it was hypothesized that endogenous steroids may inhibit EROD activities in an uncompetitive manner causing a reduction in Km
. This, however, should be confirmed. The apparent Km
values for low-affinity component of EROD in the present study were fairly similar with those reported for EROD in porcine liver microsomes by Messina et al
]. Interestingly, the results on kinetic parameters for MROD differed markedly in those two studies. Messina et al
] demonstrated that Km
value (2.2 μM) for MROD in liver microsomes was higher compared to that for EROD (0.64 μM), and compared to the Km
values (0.02 and 0.14 μM) found here (Table 3
Eadie-Hofstee plot of MROD activities was monophasic within the studied concentration range in castrated male pigs, and tended to be monophasic in entire male pigs (Figure 3
). Similarly, the kinetics for MROD activities were monophasic in human, monkey, mouse and rat [10
3.4. Inhibition of EROD and MROD activities by α-naphthoflavone (ANF), ellipticine, furafylline
ANF is generally known as a competitive inhibitor of CYP1A activity in human [17
], fish [18
] and other species. EROD and MROD activities here were inhibited in a concentration-dependent manner by ANF and ellipticine (Figures 4
). In the presence of 20 μM ANF, EROD and MROD activities decreased to 19–23% (EROD) and to 30% (MROD) of the control values. In the presence of 20 μM ellipticine, which is a specific inhibitor of human CYP1A1 activities [13
], EROD and MROD decreased to 8–10% (EROD) and to 14–16% (MROD) of the control values. In some studies on pigs, EROD was used as an indicator of CYP1A1 activities, and MROD as CYP1A2 activities [5
]. The fact that MROD activities in the present study were inhibited by a CYP1A1 inhibitor implies the lower selectivity of porcine CYP1A1/2 in catalyzing MROD activities. Alternatively, it might indicate that inhibitors, considered to be specific for human CYP1A isoforms, would not necessary inhibit the corresponding isoform in pigs. This suggestion is in line with observation of monophasic pattern of Eadie-Hofstee plots of MROD. Additionally, a monophasic Eadie-Hofstee plot does not necessarily indicate the involvement of a single enzyme; involvement of several enzymes with similar Km
values would also result in a monophasic Eadie-Hofstee plot.
Messina et al
] showed an inhibition of EROD activities by ANF and ellipticine using pig and human recombinant CYP1A2. It was demonstrated that the expression of protein CYP1A2 was positively correlated with MROD, but not EROD activities [6
]. Furafylline, a specific inhibitor of human CYP1A2, inhibited MROD activities (Figure 5
), but did not affect EROD activities (Table 4
). This inhibition of MROD, however, was weaker compared with inhibition by ANF and ellipticine. As much as 100 μM furafylline was needed to reduce MROD activities to 55–61% of the control values. It should be emphasized that specificity of furafylline as inhibitor of human CYP1A2 was investigated using activity of phenacetin O
-deethylase as an indicator of CYP1A2 activity [19
], whereas here we used 7-methoxyresorufin O
Based on the lack of inhibition of EROD activities by furafylline, it can be suggested that EROD is not a marker for CYP1A2 in porcine liver. However, the biphasic pattern of Eadie-Hofstee plots of EROD reflects the involvement of multiple enzymes, suggesting that 7-ethoxyresorufin is not a specific substrate for CYP1A1 activity in pigs. Thus, final conclusion must await isolation and characterization of porcine CYP1A1. The question whether the second EROD isozyme in porcine microsomes is a CYP1A2, remains to be answered.
No inhibition of MROD activity by furafylline was observed when no pre-incubation of microsomes and furafylline with NADPH was performed (data not shown). This observation is consistent with results from previous studies on the mechanism of furafylline inhibition of MROD. Ueng et al
] demonstrated that pre-incubation of mouse microsomes and furafylline with an NADPH-generating system resulted in strong inhibition of MROD activity. In contrast, Schultz et al
] found no differences when furafylline was incubated with microsomes from marmosets and NADPH before addition of substrate, or when furafylline and substrate were incubated together before the addition of NADPH. Inhibition constants (Ki
) of the inhibitors on EROD and MROD are shown on Table 4
. It is unclear whether the observed differences in Ki
between two groups of microsomes are due to the presence of endogenous steroids in the liver from entire male pigs or some other unknown factors. The gender-related difference in EROD and MROD activities in porcine liver microsomes was recently established [5
]. Further studies on the effect of high levels of testicular steroids on EROD and MROD activities are in progress in our laboratory.