Organic Chemicals Adsorbed onto Diesel Exhaust Particles Directly Alter the Differentiation of Fetal Thymocytes Through Arylhydrocarbon Receptor but Not Oxidative Stress Responses

Figures & data

TABLE 1 List of primers used for semi-quantitative RT-PCR

Gene Symbol	Accession number	Primer sequence (5′-3′)	PCR cycle number	Annealing temperature (°C)	Product size (bp)
Actb	NM007393	CCTCTATGCCAACACAGT AGCCACCGATCCACACAG	18	60	153
Cyp1a1	K02588	CCATGACCGGGAACTGTGG TCTGGTGAGCATCCTGGACA	26	60	344
Cyp1b1	X78445	GGACAAGGACGGCTTCATTA ACTGATGAGCGAGGATGGAG	27	60	134
Hmox1	NM010442	GTTCCCTCACCAAAAGCACA CACCCACCCCTCAAAAGATAG	23	60	324
Lgals3	X16834	AGGAGAGGGAATGATGTTGCC GGTTTGCCACTCTCAAAGGG	22	60	143
Mmp13	X66473	GCCATTACCAGTCTCCGAGGA GGAATTTGTTGGCATGACTCTCAC	28	64	534
Nqo1	NM008706	AAGAGGAGCAGAAGAAGAACAA TCGTGGAGCAAAGGAAAAA	25	60	187
Scin	U04353	GTGCTTCTAAGCATTTCCCC GAGTGAATGGCATCCAAGTG	25	60	121
Tiparp	AW120868	AGAGCTTGGTATCGGAAGGA GAATGTGCTGTTGCCTCAGT	25	60	125

TBALE 2 Up- and down-regulated gene in mouse fetal thymus exposed to DEP extracts

				Signal log ratio
Time (h)	Gene symbol	Accession symbol	Change call	Exp.1	Exp.2	Functions
6	Cyp1a1	K02588	I	5.2	5.1	Drug metabolism
	Tiparp	AW120868	I	2.2	2.5	ADP ribosylation
	Cyp1b1	X78445	I	1.8	1.7	Drug metabolism
	Lgals3	X16834	I	1.4	1.6	Cell adhesion
	Scin	U04353	I	1.6	1.2	Actin binding
	Mmp13	X66473	I	1.2	1.3	Proteolysis
24	Cyp1a1	K02588	I	4.4	4.9	Drug metabolism
	Mmp13	X66473	I	2.1	2.5	Proteolysis
	Cyp1b1	X78445	I	1.5	1.9	Drug metabolism
	Lgals3	X16834	I	1.3	1.8	Cell adhesion
	Tiparp	AW120868	I	1.7	1.7	ADP ribosylation
	Ly6a	X04653	I	1.3	1.6	Cell surface antigen
	Scin	U04353	I	1.2	1.1	Actin binding
	Cxcl9	M34815	I	1.1	1.1	Immune response
	Arg1	U51805	D	−1.2	−1.3	Hydrolysis

Fetal thymus was exposed to DEP extracts for 6 or 24 hr. Total RNA was isolated and gene expression was analyzed using an Affymetrix GeneChip system. These genes showed at least a 2-fold change in expression in both of two independent experiments. By comparison analysis using GCOS software, change call (increase (I) or decrease (D)), and signal log ratio in each two-independent experiment was calculated. Functions are from GO annotation〈http://geneontology.org/〉.

FIG. 1 Venn diagram of DEP extracts and AhR ligand (TCDD and B[a]P)-regulated genes. Fetal thymus was exposed to DEP extracts, TCDD, or B[a]P for 24 hrs. Total RNA was isolated and gene expression was analyzed using an Affymetrix GeneChip. These genes showed at least a 2-fold change in expression in both of two independent experiments, compared to controls.

FIG. 2 Confirmation of gene chip data in fetal thymuses exposed to DEP extracts, TCDD, and B[a]P using semi-quantitative RT-PCR. Fetal thymus was exposed to DEP extracts, TCDD, or B[a]P for 24 hrs, and ds-cDNAs obtained in two independent experiments were subjected to semi-quantitative PCR using each specific primer pair (Table 1). PCR products were separated in a 1.2% SYNER gel containing 0.5 μg/ml ethidium bromide.

FIG. 3 Venn diagram of DEP extracts and quinones (NPQ and PHQ)-regulated genes. Fetal thymus was exposed to DEP extracts, NPQ, or PHQ for 24 hours. Total RNA was isolated and gene expression was analyzed using an Affymetrix GeneChip. These genes showed at least a 2-fold change in expression in both of two independent experiments, compared to controls.

FIG. 4 Confirmation of gene chip data in fetal thymuses exposed to DEP extracts, NPQ, and PHQ using semi-quantitative RT-PCR. Fetal thymus was exposed to DEP extracts, NPQ, or PHQ for 24 hrs, and ds-cDNAs obtained in two independent experiments were subjected to semi-quantitative PCR using each specific primer pair (Table 1). PCR products were separated in a 1.2% SYNER gel containing 0.5 μg/ml ethidium bromide.

FIG. 5 Measurement of the mRNA expression of stress response genes using semi-quantitative RT-PCR. Ds-cDNAs of stress response genes (Nqo1 and Hmox1) were amplified using semi-quantitative PCR and visualized using ethidium bromide. Ds-cDNAs were identical to the samples used in the Affymetrix GeneChip analysis (Figure 1 and Figure 3).

FIG. 6 Hierarchical clustering analysis of the genes that were either up- or down-regulated by a 24 hr exposure to DEP extracts or their constituents. Values obtained from Affymetrix GeneChip analysis were normalized using the mean of the vehicle-treated controls (cream color), and hierarchical clustering was performed using GeneSpring. The results are presented as the mean of ratio of the signal intensities from two independent experiments. Up-regulated genes appear red while down-regulated genes appear blue.

FIG. 7 Effects of DEP extracts or their constituents on thymocyte cellularity. Fetal thymus was exposed to DEP extracts or the described chemicals for 4 days and thymocyte cell numbers (A) and subset populations (B) were examined. The data are expressed as the mean percent of control ± SEM (n = 3), compared to control. Asterisk indicates statistically significant difference (p < 0.05) from the corresponding control.