Liver enzymes in White Leghorns selected for the sheep red blood cell immune response

Liver enzymes in White Leghorns selected for the sheep red blood cell immune response S. Blevins,* P. B. Siegel,* D. J. Blodgett,† M. Ehrich,† and R. M. Lewis*1 *Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg 24061; and †Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061 Liver tissue was analyzed for quinone reductase, glutathione-S-transferase, and cytochrome P450 3A4 activity. All data were analyzed using ANOVA. There were no consistent differences in enzyme activity between high- and low-antibody lines at any age. Cytochrome P450 3A4 activity was substantially greater in 4- and 8-wk than in 12- and 20-wk-old chickens (P < 0.001). This study provides insights into enzyme activities of liver enzymes; however, except for cytochrome P450 3A4, no clear trends across ages were observed.

Key words: chicken, enzyme activity, liver 2012 Poultry Science 91:322–326 doi:10.3382/ps.2011-01764

INTRODUCTION

der of the individual’s life (Hines, 2007). Unlike humans, male mice express appreciable levels of CYP450 3A4 before birth, increase this expression rapidly after birth, and begin to decline this expression by 45 d of age (Hart et al., 2009). Furthermore, there is considerable variability within age groups in CYP450 3A4 enzyme expression due to genotype. Some authors report times of hypervariability in enzyme expression between individuals of the same age (Hines, 2007). This variability is usually attributed to structural polymorphisms (Hines and McCarver, 2002). For example, humans who are homozygous for the QR gene, NQ01, completely lack QR enzyme activity, whereas enzyme activity varies among heterozygous individuals (Talalay and Dinkova-Kostova, 2004). Enzyme activity also varies in chickens. Coulet et al. (1996) reported that GST activity was constant in chickens between 3 and 9 wk of age. However, by 12 wk, GST activity increased by 60%. Maurice et al. (1991) found male Barred Plymouth Rock chickens had approximately 15% more hepatic GST activity than that of the females. Chicks have been reported by Lorr and Bloom (1987) to have detectable cytochrome activity as early as d 7 of incubation. This cytochrome activity increased dramatically at the day of hatch and reached a plateau 3 d posthatch (Lorr and Bloom, 1987). The objective of this study was to characterize age and line differences in GST, QR, and CYP450 3A4 ac-

Enzymes produced by the liver are essential for xenobiotic and pharmaceutical metabolism. Glutathione-Stransferase (GST) and quinone reductase (QR) catalyze reactions that make xenobiotics more polar so that they can be easily excreted in bile (Ioannides, 2002). Furthermore, enzymes of the cytochrome (CY) P450 3A family are responsible for 45 to 60% of pharmaceutical metabolism (Burk and Wojnowski, 2004). Activity of metabolic enzymes is considered to be heavily dependent upon species, sex, age, genotype, and environment (Wauthier et al., 2007; Waxman and Holloway, 2009). Changes in enzyme activity during development determine drug efficacy as well as toxicity of compounds. Therefore, it is important to understand how enzyme activity changes over time, and differs between species and genotypes, to understand how individuals cope with toxicants over their lifetimes. Cytochrome P450 3A4 expression is nearly devoid in the human fetus. It is not until approximately 3 yr of age that CYP450 3A7 expression ceases and CYP450 3A4 becomes the dominant cytochrome for the remain©2012 Poultry Science Association Inc. Received June 28, 2011. Accepted October 27, 2011. 1 Corresponding author: [email protected]

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ABSTRACT Liver enzymes are essential to xenobiotic metabolism. Expression of these enzymes is dependent upon factors such as age and sex. The objective of this study was to determine basal liver enzyme levels in male and female White Leghorn chickens to provide reference values for future studies. Chickens from 2 lines divergently selected for 35 generations for high antibody and low antibody immune response to SRBC were used. Six male and 6 female chickens from each line were killed at each of 4, 8, 12, and 20 wk of age. Livers were collected and used for enzyme analyses.

RESEARCH NOTE

tivity between 2 lines of White Leghorn chickens divergently selected for humoral immune response. This information will provide baseline enzyme activity that can be used for comparison in future toxicological studies.

MATERIALS AND METHODS Birds and Tissue Collection

Enzyme Activity Assays Sample Preparation. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Liver samples were weighed and a 25% homogenate was prepared in 0.1 M phosphate buffer (pH 7.4 with 1.15% KCl) using a Polytron blender (Brinkman Instruments, Westbury, NY). Homogenates were centrifuged for 10 min at 15,000 × g at 5°C. The supernatant was recentrifuged for 60 min at 50,000 × g at 5°C. The supernatant, cytosol, was transferred and stored at −80°C until assayed. The pelleted portion, containing microsomes, was resuspended in an equal volume of 10 mM Tris-acetate

buffer (pH 7.4, 0.1 M EDTA, 20% glycerol) and stored at −80°C. Enzyme activity and protein assays were performed in 96-well microtiter plates. Quinone reductase and GST activities were measured in liver cytosol fractions and CYP450 3A4 activity was measured in microsomal fractions. For each enzyme assay and the protein assay, samples from each chicken were analyzed in triplicate. QR. Quinone reductase activity was assayed using a protocol described by Prochaska and Santamaria (1988). Quinone reductase activity is indirectly determined by measuring the amount of NADPH oxidized (nmol) per minute per milligram of protein. Liver cytosol was diluted 1:8 with 0.1 M phosphate buffer (pH 7.4, 1.15% KCl). Fifty microliters of diluted cytosol and 200 μL of the reaction mixture were added to each well. After 5 min, the reaction was stopped with a 0.3 mM dicoumarol solution. The plate was read at 610 nm using a microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA). The concentration of enzyme was determined using an extinction coefficient of 11,300 M/cm and path length of 0.57 cm. Enzyme activity (nmol of NAPDH oxidized/min per mg of protein) was calculated. GST. A 20 mM 1-chloro-2,4-dinitrobenzene substrate and a 20 mM glutathione cofactor were used to assay GST activity. Liver cytosol was diluted 1:32 with 0.1 M phosphate buffer (pH 7.4, 1.15% KCl). Each well contained 40 μL of diluted cytosol, 220 μL of 0.1 M sodium phosphate buffer (pH 6.5) warmed to 30°C, 20 μL of 1-chloro-2,4-dinitrobenze solution, and 20 μL of glutathione solution. The plate was read at 340 nm using a microplate spectrophotometer (Molecular Devices Corp.) for 5 min with readings taken at 15-s intervals. An extinction coefficient of 9.6 mM/cm and a light path length of 0.57 cm were used to calculate the enzyme concentration. Enzyme activity was expressed in micromoles of glutathione conjugated/min per milligram of protein (Habig et al., 1974; Kaplowitz et al., 1975; Mannervik and Jemth, 1999). CYP450 3A4. Cytochrome P450, specifically the 3A4 family, activity was determined using a fluorescent spectrophotometry method derived from Crespi et al. (1997). The substrate was a solution of 400 μM 7-benzyloxyquinoline (7-BQ) in 50 mM phosphate buffer (pH 7.4). Dilutions of 400 μM 7-BQ were made to create a 0 to 40 μM 7-BQ standard curve. One hundred microliters of the respective standards or 100 μL of the 80 μM 7-BQ substrate was added to each of the microplate wells. The plate was incubated at 37°C for 15 min. Opaque 96-well plates were used to prevent background interference. The microsomal fraction of each sample was diluted 1:4 with 10 mM Tris-acetate buffer (pH 7.4, 0.1 mM EDTA, 20% glycerol). Following the 15-min incubation, 20 μL of each diluted microsomal sample was added to the wells. Eighty microliters of the NADP-generating system, as described by Crespi et al. (1997), was added to each well. The reaction progressed for 30 min at

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Selection lines of White Leghorn chickens have been developed at Virginia Tech. These lines have been selected for 35 generations based on antibody response to a 0.1-mL injection of SRBC 5 d postinjection. Through this selection, 2 lines have been developed: a low antibody (LA) and a high antibody (HA) response line (Gross and Siegel, 1980; Martin and Dunnington, 1990; Kuehn et al., 2006). In the design, S34 chickens from each line were supposed to be from a single hatch and grown to 4 ages: 4, 8, 12, and 20 wk. However, a limited number of chicks was available from the initial hatch. Therefore, chicks from a second hatch were also used. Four-, 12-, and 20-wk-old females, as well as 4- and 8-wk-old males, hatched March 20, 2007; 12- and 20-wk-old males hatched August 13, 2007. Four- and 8-wk-old chickens from these lines were housed in 3.7 × 1.6 m floor pens and allowed free access to water and a mash diet (corn-soybean meal: 14% CP; 2,827 kcal of ME/kg on a DM basis). They were exposed to 24L:0D from the day of hatch to d 7, 23L:1D from d 7 to 6 wk, and 14L:10D thereafter. Twelve- and 20-wk-old birds were housed the same as the 4- and 8-wk-old chickens until they were 12 wk old. At 12 wk, they were moved to 5.2 × 4.6 m floor pens. Six male and 6 female chickens from both lines were killed by cervical dislocation at each age. A section of the left lobe of the liver was removed, chopped into small (approximately 3-mm) pieces using a razor blade, and split into 3 samples. The samples were doublewrapped in aluminum foil and frozen in liquid nitrogen. Samples were stored at −80°C. The Virginia Tech Institutional Animal Care and Use Committee approved all housing conditions and experimental procedures.

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Table 1. Least squares means for quinone reductase, glutathione-S-transferase, and cytochrome P450 3A4 for male and female chickens by line and age (wk) Quinone reductase (nmol of NADPH oxidized/min per mg of protein)2 Line1

4 wk

8 wk

12 wk

20 wk

Male

HA LA SEM HA LA SEM

2.78a 2.24a 0.20 3.03c 2.69c 0.18

2.79a 2.14a 0.20 2.71c 2.48c 0.18

1.65a 2.06a 0.20 3.01c 2.22c 0.18

2.73a 2.30a 0.20 1.69c 2.38c 0.18

Female

           

Cytochrome P450 3A4 (µmol of 7-BQ4 oxidized/min per mg of protein)

4 wk

8 wk

12 wk

20 wk

4 wk

8 wk

12 wk

20 wk

0.102b 0.074a 0.006 0.083c 0.084c 0.005

0.079a 0.087a 0.006 0.091c 0.087c 0.005

0.066a 0.069a 0.006 0.092c 0.091c 0.005

0.082a 0.084a 0.006 0.091c 0.084c 0.005

3.06a 3.24a 0.30 3.79c 2.94c 0.31

3.34a 3.65a 0.30 3.21c 3.16c 0.31

1.27a 1.12a 0.30 2.53c 2.79c 0.31

0.628b 0.715b 0.30 1.11d 1.21d 0.31

           

a,bMeans

among males (across lines) with different superscripts are different (P < 0.05). among females (across lines) with different superscripts are different (P < 0.05). 1HA = high antibody; and LA = low antibody. 2For each sex, a line by age interaction was detected (P < 0.05). 3For males, a line by age interaction was detected (P < 0.001). This was due to the difference between lines at 4 wk. 47-Benzyloxyquinoline. c,dMeans

37°C and was stopped with an 80% acetonitrile and 20% 0.5 M Tris buffer (pH 9) solution. The plate was read at an excitation and emission wavelength of 410 nm and 538 nm, respectively (Spectra Max M5, Molecular Devices Corp.). Enzyme activity was determined via interpolation from the standard curve. Activity was reported as micromoles of 7-BQ oxidized/min per milligram of protein. Protein. All enzyme activities are reported on a per milligram of protein basis. Cytosolic as well as microsomal proteins were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). This is a protein-dye binding method based on the Bradford method (Bradford, 1976). Bovine serum albumin was used to create a standard curve.

Statistical Analysis All analyses were conducted using SAS 9.1 (SAS Institute Inc., Cary, NC). Data on male and female chickens were analyzed separately using the GLM procedure. The model fitted was Yijk = µ + Ai + Lj + (AL)ij + eijk, where Yijk was the enzyme activity for a chicken (k = 1,…,6) at age A (i = 1, …,4, for 4, 8, 12, and 20 wk, respectively) from selection line L (j = 1 or 2, for HA or LA, respectively), with (AL)ij as the age by line interaction and eijk the residual error. The overall mean was µ. Least squares means for age and line were compared using the Student’s t-test.

RESULTS Least squared means for QR, GST, and CYP450 3A4 for male and female chickens are provided in Table 1. These results are shown by line and age.

QR Males. There was a difference in QR activity across ages in male chickens (P < 0.01). This difference was largely due to a drop in QR activity at 12 wk, at which time, HA chickens had slightly lower QR activity than that of LA chickens (P < 0.05). Because of a subsequent rise in QR activity at 20 wk, an age by line interaction was detected (P < 0.05). Females. Quinone reductase activity was lower in female chickens at 20 wk compared with that at 4, 8, and 12 wk chickens (P < 0.001). There was also an age by line interaction (P < 0.001). Unlike in males, in females at 12 wk of age, HA chickens had higher activity than LA chickens (P < 0.001). However, at 20 wk (P < 0.05), LA chickens had higher activity.

GST Males. Glutathione-S-transferase activity was much lower at 12 wk compared with 4, 8, and 20 wk in males (P < 0.001). There was also a strong age by line interaction (P < 0.001) because of the difference between lines at 4 wk. At this one age, HA chickens had higher activity than that of LA chickens. Females. There were no differences in GST activity among ages (P = 0.354) or lines (P = 0.526) in females. Furthermore, there was no interaction between age and line for female chickens (P = 0.620).

CYP450 3A4 Males. Cytochrome P450 3A4 activity was fairly constant in males from 4 to 8 wk averaging 3.32 μmol of 7-BQ oxidized/min per milligram of protein. At 12 wk, activity declined to 1.19 μmol of 7-BQ oxidized/ min per milligram of protein. This decline continued at 20 wk with an average of 0.67 μmol of 7-BQ oxidized/ min per milligram of protein (P < 0.001). There was no

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Sex

Glutathione-S-transferase (µmol of glutathione conjugated/min per mg of protein)3

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GST

effect of line (P = 0.602) or age by line interaction (P = 0.850; Figure 1). Females. In females, CYP450 3A4 activity changed with age (P < 0.001). At 4 and 8 wk, activity remained constant, averaging 3.27 µmoles of 7-BQ oxidized/min per mg protein. Activity declined at 12 wk to 2.66 μmol of 7-BQ oxidized/min per milligram of protein and further declined to 1.16 μmol of 7-BQ oxidized/min per milligram of protein at 20 wk. There was no line effect (P = 0.551) or age by line interaction (P = 0.350; Figure 1).

DISCUSSION QR There is a dearth of information in the literature for chickens on patterns of QR activity across ages, with patterns inconsistent between sexes and lines in this study. In humans, QR activity seems to be heavily influenced by genotype at the NQ01 locus (Talalay and Dinkova- Kostova, 2004). An epidemiological study in China found that industrial workers with a mutant NQ01 allele were more likely to develop hemotoxicity due to benzene exposure than those with normal NQ01 alleles (Nebert et al., 2002). Furthermore, the NQ01 genotype affected breast cancer survival rates (Fagerholm et al., 2008). The 5-yr survival rate of women homozygous for the NQ01 mutant allele, NQ01*2, was 47% compared with 67% in women with a normal NQ01 genotype. The NQ01*2 genotype was associated with reduced QR activity. It can be hypothesized that chickens with lower QR activity had different variants of the NQ01 genotype.

CYP450 3A4 Cytochrome P450 3A4 activity was the only enzyme that had a clear trend over time: activity was high in young chickens but then decreased substantially with age (Figure 1). Hart et al. (2009) found that CYP450 3A4 gene expression increased rapidly in young mice and then decreased between 20 and 45 d of age. Coulet et al. (1996) found that CYP450 3A4 protein expression increased in male broilers from 3 to 9 wk of age, but its expression decreased after 12 wk. Those results are consistent with this study, where CYP450 3A4 activity in males declined appreciably at 12 wk. The higher CYP450 3A4 activity in younger chickens may reflect that it was the more dominant cytochrome at that developmental stage. For instance, younger individuals may need such heightened CYP450 3A4 activity to cope with environmental challenges early in life before other physiologically processes mature. Evidence in humans supports that supposition. Cytochrome P450 3A7 is the dominant cytochrome in young children, but as they age, its activity wanes and CYP450 3A4 becomes dominant (Hines, 2007).

ACKNOWLEDGMENTS We thank C. Honaker for technical (laboratory) support; M. Graham, J. Thomas, and B. Duncan for flock husbandry; and E. Stephens for technical editing (Virginia Tech, Blacksburg). We also thank several undergraduate research students for their assistance, particularly P. Hickey, R. Shores, and W. Martin (Virginia Tech).

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Figure 1. Cytochrome P450 3A4 activity across lines for male and female chickens. Cytochrome P450 3A4 activity declined over time in both males (P < 0.001) and females (P < 0.001). 7-BQ = 7-benzyl­ oxyquinoline.

With the exception of 4-wk-old males, GST activity was relatively similar between sexes and lines and across ages. Maurice et al. (1991), however, found distinct sex differences in hepatic GST activity between Barred Plymouth Rock and Rhode Island Red chickens at 30 wk of age. In their study, male chickens had significantly higher GST activity than females. They also found breed differences. The Barred Plymouth Rocks had significantly higher GST activity than that of the Rhode Island Reds. Thus, at older ages than those in the current study, sex and breed differences may exist. Differences in enzyme activity among ages have been observed in rats for GST. Jang et al. (2001) reported hepatic GST activity gradually increased with age in Wistar rats. Such was not the case in the current study, as there was no consistent pattern in GST activity across ages. This finding agrees with that of Jung and Henke (1996), who found no clear pattern in GST activity in 1-, 2-, 3-, 5-, and 12-mo-old rats. Perhaps, as Jung and Henke (1996) concluded, there is a more complicated network of interactions involved in enzyme activity than simply age.

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