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Abstract

Exposure to endocrine-disrupting chemicals (EDCs) and maternal endogenous estrogen may cause hypospadias, common congenital anomaly. Several organochlorine pesticides (OCPs) have been reported to possess an endocrine-disrupting potential. Cytochrome P4501A1 (CYP1A1) and glutathione S-transferases (GSTM1 and GSTT1) of xenobiotic metabolizing enzyme family are involved in the metabolism of various environmental toxicants and steroidal hormones. Hence, the present study was designed to evaluate the role of CYP1A1, GSTM1, GSTT1 genes polymorphism, OCPs levels and risk of hypospadias. A total of 80 hypospadiac and 120 age-matched control boys were included. OCP levels in blood were determined using Gas Chromatograph equipped with electron capture detector (GC-ECD) and polymorphism in CYP1A1, GSTM1 and GSTT1 genes was evaluated by RFLP and multiplex PCR method. We observed significant high levels of β-hexachlorohexane (HCH), γ-HCH, and p,p′-dichlorodiphenyl-dichloroethylene (p,p’-DDE) in the cases. CYP1A1 polymorphisms were not significantly different among cases and controls, whereas concomitant deletion of GSTM1 and GSTT1 genotypes was significantly higher in cases as compared to controls. However, after adjusting for low birth weight and maternal occupational exposure, the results did not remain significant but odds of risk was higher (OR = 1.72, p = 0.14) among cases. In conclusion, our study suggests irrespective of genetic predisposition, higher level of some OCPs may be associated with increased risk of hypospadias.

Keywords

hypospadias polymorphism CYP1A1 GSTM1 GSTT1 pesticides levels

Introduction

Hypospadias, a common congenital anomaly, is the incomplete fusion of the urethral folds, resulting in a urethral opening on the ventral surface of the penis or on the scrotum or the perineum and probably a complex disorder caused by both genetic and environmental influences, either alone or in combination.¹ It has been hypothesized that in-utero exposure to environmental factors such as endocrine-disrupting chemicals (EDCs) with estrogenic or anti-androgenic activity or maternal endogenous estrogen affecting androgen homeostasis during fetal life may cause hypospadias.^2–4

EDCs are exogenous substances that alter functions of the endocrine system and adversely affect reproductive health and fetal development.^5,6 Organochlorine pesticides (OCPs) such as hexachlorohexane (HCH), p,p′-dichlorodiphenyl-dichloroethylene (p,p′-DDE) p,p’-dichlorodiphenyl-trichloroethane (p,p′-DDT) and dieldrin have been reported to possess an endocrine-disrupting potential.^7–9 In India, these pesticides have been banned for use in agriculture but are still in use for public health programmes.¹⁰ OCPs are lipophillic in nature, having half-lives from months to years and biomagnifies through food chain. As a result, OCPs can still be detected in ecosystem i.e. in water, soil, air, food items etc., creating a persistent exposure to human and tend to accumulate in adipose tissue. In recent studies from our laboratory, we have reported an association between OCPs levels such as HCH, DDT, DDE as well as endosulfan-α and β with preterm birth and recurrent miscarriages. Placental transfer of these endocrine disruptors from the mother to fetus has been reported, which indicates the transfer to unborn is dependent on maternal daily exposure as well as accumulated maternal body burden which may affect growth and development of the baby in the womb.^8,11–14

In human system, detoxification of xenobiotic substances is carried out in two phases. In phase I, the reactive intermediates are produced and in phase II, these reactive intermediates are conjugated with polar groups like glutathione. Cytochrome P 450 (CYP1A1) is an important phase I metabolizing enzyme, which plays a vital role in metabolism of environmental chemicals and also catalyzes the hydroxylation of endogenous steroids including estradiol, progesterone, pregnenolone etc.^15,16 Metabolites of estrogens or other hormones through CYP1A1 might be critical for developing hypospadias, because normal urethral development depends on the delicate balance of these hormones.¹⁷ CYP1A1 gene consists of four functional polymorphism. CYP1A1 m1 (MspI) is T→C transition located downstream of exon 7,^18,19 it does not exert any effect on CYP1A1 induction²⁰ but increases the microsomal enzymatic activity.²¹ CYP1A1 m2, A→G transition leads to an amino-acid substitution of valine (Val) for isoleucine (Ile) in exon 7,¹⁸ significantly associated with CYP1A1 inducibility.^21,22 CYP1A1 m3 has mutation in exon 7 and is reported to be African American-specific.²⁰ CYP1A1 m4 polymorphism is located two base pair upstream of m2 site leading to an amino acid substitution of threonine (Thr) for asparagines (Asn) in the heme-binding region.¹⁸ However, effect of this polymorphism on activity of enzyme is not yet clearly elucidated.

Glutathione S-transferases (GSTs) are important phase II metabolic enzymes that detoxify metabolic intermediates by conjugating them with glutathione.²³ The GSTM1 gene is located on chromosome 1 and 20%–50% of individuals do not express this enzyme due to a homozygous deletion, known as GSTM1*0/0 or null allele,²⁴ whereas GSTT1 gene is located on chromosome 22 and 20%–60% of individuals do not express the enzyme, also due to gene deletion, known as the GSTT1*0/0 allele. Inherited homozygous deletion of these genes has been demonstrated to result in lack of its enzymatic activity.²⁵ There are ethnic differences in the frequency of genetic polymorphism in biotransformation enzymes.^26–28

Few studies have suggested that there may be a possible association between OCPs levels and polymorphism of these metabolic susceptibility genes with increased/decreased risk of hypospadias but are not clearly evaluated. Hence, the present study was designed to evaluate the role of CYP1A1, GSTM1, GSTT1 gene polymorphism and OCPs levels with the increased/decreased risk of hypospadias in north Indian population.

Materials and methods

Study population

Present case-control study was performed at Department of Biochemistry, UCMS & GTB Hospital. Eligible cases were male children diagnosed with hypospadias without concomitant malformation (cleft lip and/or plate or cryptochidism) and chromosomal abnormalities at the Department of Pediatrics, UCMS & GTB Hospital and Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India. In this study, 80 hypospadias cases diagnosed and confirmed by a pediatric urologist and randomly selected 120 age-matched (1–10 years) healthy controls boys not suffering from any clinically detectible illness were included. A self-administered questionnaire was used to obtain relevant information including maternal age, maternal history of environmental/occupational exposure and dietary habits (vegetarian / non-vegetarian). Information about birth weight was collected through infant medical records (Table 1 ). Informed consent was obtained from parents/guardians of all the subjects and the study was approved by Institutional Ethics Committee for Human Research. A total of 3 mL peripheral blood sample was collected in EDTA vial; 1 mL of blood sample was used for OCPs analysis and from rest of the blood sample, DNA was isolated for further genotyping. All the experiments were performed within 12 hours of sample collection. Genomic DNA was extracted by QIAamp Blood DNA mini kit (Qiagen, Valencia, CA, USA) following manufacture’s protocol.

Table 1.

Characteristics of case mothers (80) who had hypospadiac boys and control mothers (120) who did not

		Case No	Control No	OR	95%CI	p-value
Low birth weight (<2500 g)	Yes	31	22	2.81	(1.478-5.372)	0.002^a
	No	49	98	1.00
Maternal age at delivery ≥30	Yes	32	52	0.87	(0.490-1.549)	0.639
	No	48	68	1.00
Maternal h/o occupational/ environmental exposure	Yes	29	18	3.22	(1.637-6.344)	0.001^a
	No	51	102	1.00
Dietary information	Vegetarian	52	71	1.28	(0.713-2.303)	0.407
	Non-Vegetarian	28	49	1.00

^a Statistically significant p < 0.05.

Extraction, cleanup and quantification of OCPs from blood samples

All the chemicals used in the process were of high-purity grade. The HPLC-grade solvents were checked for any contamination before OCPs extraction. OCPs were extracted according to method of Bush et al.²⁹ Blood sample was taken in a 50 mL conical flask for extraction. Hexane and acetone were added in 2:1 ratio and the contents were shaken well at room temp for 30 minutes. The extract was then centrifuged for 10 minutes at 2000 rpm and supernatant was collected in a clean test tube. The remaining portion was again extracted twice using the same process. Cleanup of solvent fractions was done by column chromatography following USEPA method 3620B. Elute was collected in 100 mL beaker and evaporated to concentrate the sample. The concentrated residues were dissolved in hexane for further analysis.

Quantification of OCP levels (HCH, aldrin, dieldrin, endosulfan α, endosulfan β, DDT and DDE)was done by Perkin Elmer GC equipped with ⁶³Ni selective electron capture detector. The column used was Elite-GC DB-5, 60 m and 0.25 mm ID. The carrier gas and make-up gas was nitrogen, with a 2 mL/min and 35 mL/min flow rate, respectively, employing the split-less mode. Final extract (1 µL) was injected at a temperature of 170°C with a hold time of 1 minute. The temperature was raised from 170°C to 225°C at a rate of 5°C/min, with a hold time of 5 minutes and finally from 225°C to 275°C at a rate of 6°C/minutes, with a hold time of 15 minutes. The total run length was 40 min/sample.

Quantitative analysis of OCPs in each sample was done by comparing the peak heights with those obtained from a chromatogram of a mixed OCPs standard (Supelco, Sigma-Aldrich, Saint Louis, USA) of known concentration. The detection limit of the detector was <0.05 pg perchloroethylene with nitrogen as a carrier gas. The detection limit of the method was 4 picogram/mL (pg/mL) for each OCP. For quality control process, 10 blood samples in triplicate were spiked with a mixed standard of OCPs at 5 and 25 ng/mL. The average recoveries of fortified samples exceeded 95%. The case and control samples were run in the same analytical batches and further, a quality control check of the samples was always run with each set of samples to maintain accuracy.

Genotyping

CYP1A1 genotyping

For analysis of CYP1A1 polymorphism at m1 and m4 site, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method was used, whereas, for m2 polymorphism an allele-specific PCR method was used. For CYP1A1 m1 polymorphism a 50 μL reaction mixture consisted of ~50 ng of genomic DNA, 10 μM of each primer, 0.2 mM of dNTPs mixture (Bangalore Genei, India), 1.5 mM of MgCl₂, 1.5 unit of Taq polymerase with 1 × PCR reaction buffer (Bangalore Genei, India). For analysis of CYP1A1 m2 polymorphism, PCR amplification was done in 50 μL of reaction mixture consisted of ~100 ng of the template DNA, 10 μM of each primer, 0.2 mM of dNTPs mixture, 1.5 mM of MgCl₂, 1.0 unit of Taq polymerase with 1 × PCR reaction buffer. PCR reaction condition for m4 allele was same as for m1 allele as recently reported from our laboratory.³⁰

GSTM1 and GSTT1 genotyping

GSTM1 and GSTT1 genotyping was carried out with multiplex PCR method of Abdel-Rahman et al.,³¹ using three sets of primers. Multiplex PCR reaction was performed in 25 µL reaction mixture consisting of ~200 ng of DNA, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂ (pH 8.4), 0.5 mM of each dNTPs, 0.3 µM each forward and reverse primer and 1.5 U Taq polymerase (Bangalore Genei, India). PCR conditions, primers sequences, restriction enzymes (for RFLP methods) and length of the expected fragments (amplified and digested) to identify CYP1A1, GSTM1 and GSTT1 polymorphism are given in Table 2 . All PCR amplifications were performed in Eppendorf Mastercycler Gradient-5331 thermocycler.

Table 2.

Primers, PCR conditions, restriction enzymes/PCR method and DNA fragments to identify CYP1A1, GSTM1 and GSTT1 polymorphism^a

Primers	PCR profile	RE/PCR Method	DNA Fragments	References
CYP1A1 m1	94°C for 5 min	MspI (RFLP)	AF- 340
m1F 5’-AAGAGGTG TAGC CGCTGCACT-3’	94°C for 1 min		NH-340	Song et al. ¹⁹
m1R 5’-TAGGAGTCTCTCATGCCT-3’	61°C for 1 min		HE-340, 200,140
	72°C for 1 min		MH-200, 140
	72°C for 10 min
CYP1A1 m2
m2F 5’-GAAGTGTAT CGGTGAGACCA-3’	95°C for 5 min	AS-PCR	NA ~205	Hayashi et al.²²
m2R 5’-GAAGTGTATCGGTGAGACCG-3’	95°C for 1 min
CommR 5’-GTAGACAGAGTCTAGGCCTCA-3’	65°C for 1 min
	72°C for 1 min
	72°C for 10 min
CYP1A1 m4
m4F 5’TTCCACCCGTT GCAGCAGGATAGCC-3’	94°C for 5 min	Bsa I (RFLP)	AF -204	Cascorbi et al.¹⁸
m4R 5’-CTGTCTCC CTCTGGTTACAGG AAG-3’	94°C for 30 s		NH-139,65
	63°C for 30 s		HE- 204,139, 65
	72°C for 30 s		MH-204
	72°C for 10 min
GSTM1 and GSTT1
M1F 5-GAACTCCCTGAAAAGCTA AAG C-3’	94°C for 5 min	Multiplex	GSTT1-480	Abdel-Rahman et al.³¹
M1R 5'-GTTGGGCTCAAATATACGGTGG-3'	94°C for 2 min	PCR	CYP1A1-312
T1F 5’-'TTCCTTACTGGTCCTCACATCTC- 3'	59°C for 1 min		GSTM1-215
T1R 5’-TCACCGGATCATGGCCAGCA-3'	72°C for 1 min
Internal Control	72°C for 10 min
CYP1A1F 5-GAACTGCCACTTCAGCTGTCT-3'3'
CYP1A1R 5'-CAGCTGCATTTGGAAGTGCTC-

Abbreviations: AS-PCR: allele-specific polymerase chain reaction, AF: amplified fragment, NH: normal homozygote, HE: heterozygote, MH: mutant homozygote, RE: restriction enzymes.

^a For all RFLP methods amplified fragments were digested at 37°C for 3 hours. All restriction enzymes were purchased from New England Biolabs, Beverly, USA.

Statistical analysis

We performed statistical analysis to determine associations between OCPs, CYP1A1, GSTM1 and GSTT1 polymorphism with the risk of hypospadias. Descriptive statistics for OCPs were expressed as mean ± SD and 25th, 50th and 75th percentile and data were analyzed by unpaired ‘t’-test. We computed odds ratios (ORs) for hypospadias and 95% confidence interval (CI) by logistic regression. For gene-environment interaction, one-way analysis of variance followed by Tukey’s test and Student ‘t’ test was performed for multiple comparison. All statistical analyses were performed using SPSS 17.0 statistical software for Windows (SPSS Inc., Chicago, Illinois, USA).

Results

Demographic profile of children and mothers in both cases and controls are given in Table 1. We found a significant positive association between low birth weight (<2500g) and hypospadias (OR = 2.81, 95% CI = 1.478−5.372). The OR for women of 30 years of age or older compared with younger ones was 0.87 (95% CI = 0.490−1.549). History of occupational exposure during pregnancy appear to have a significant association with the risk of hypospadias (OR = 3.22, 95% CI = 1.637−6.344). We did not find any significant association of dietary intake (veg/non-veg) with the risk of hypospadias (OR = 1.28, 95% CI = 0.713-2.303). OCPs residue levels (ng/mL) of hypospadias boys and control samples are listed in Table 3 . We found significantly higher levels of β-HCH, γ-HCH and p,p’-DDE in cases as compared to controls.

Table 3.

Comparison of OCP Levels (ng/mL) in blood of 80 hypospadias boys and 120 control samples

	Controls (120)				Cases (80)
	Mean ± SD	25%	50%	75%	Mean ± SD	25%	50%	75%	p-value
α-HCH	4.45 ± 1.67	3.56	4.47	5.65	4.59 ± 2.03	2.78	4.52	6.07	0.609
β-HCH	3.66 ± 2.19	2.17	3.55	4.66	6.64 ± 3.75	4.02	6.00	8.49	<0.001^a
γ-HCH	2.96 ± 1.36	2.16	3.24	4.66	4.39 ± 2.62	2.34	3.98	6.25	<0.001^a
T-HCH	10.99 ± 3.29	9.01	11.30	13.14	15.63 ± 5.89	12.66	15.49	18.75	<0.001^a
Aldrin	1.65 ± 1.32	0.19	1.59	2.69	1.83 ± 1.57	0.17	1.66	2.66	0.295
Dieldrin	1.98 ± 1.62	0.48	2.08	3.02	2.30 ± 2.63	0.00	1.27	4.53	0.332
Endo I	0.96 ± 0.76	0.00	1.09	1.51	1.27 ± 1.48	0.00	1.03	2.09	0.094
Endo II	0.84 ± 0.67	0.00	1.07	1.30	0.92 ± 1.03	0.00	0.81	1.76	0.512
p,p’DDE	2.36 ± 1.8	0.45	2.36	3.78	3.31 ± 2.50	1.26	2.95	4.92	<0.005^a
p,p’DDT	1.16 ± 0.9	0.74	1.24	1.65	1.17 ±1.26	0.00	0.84	2.14	0.919

^a Statistically significant p <0.05

The frequencies of the CYP1A1, GSTM1 and GSTT1 genotypes were also compared between the subjects (Table 4 ). Heterozygous CYP1A1m1 (wt/mt) as well as homozygous CYP1A1m1 (mt/mt) were less frequently found in cases as compared to controls (OR = 0.79, 95% CI = 0.422−1.498; OR = 0.88, 95% CI = 0.409−1.933, respectively). Similar results were also observed in CYP1A1m2 polymorphism, whereas polymorphism in CYP1A1m4 was completely absent. Individual GSTM1 and GSTT1 null deletion were present in high frequencies in cases, but it was not significant (OR = 1.28, 95% CI = 0.665–2.492); OR = 1.32, 95% CI = 0.728–2.418, respectively). However, the odds of finding shows that concomitant deletion of GSTM1 and GSTT1 genotype were significantly higher in cases (OR = 2.28, 95% CI =1.139–4.599) compared to controls (Figure 1 ). After adjusting for low birth weight and occupational exposure, the ORs of CYP1A1, GSTM1 and GSTT1 genotypes did not remain significant (Table 5 ).

Table 4.

Frequencies of CYP1A1, GSTM1 and GSTT1 genotype among 80 hypospadias and 120 control boys

Genotype	Case No(%)	Control No(%)	OR	95%CI	p-value
CYP1A1 m1
wt/wt	33 (41.25)	50 (41.67)	1.00
wt/mt	31 (38.75)	52 (43.33)	0.79	(0.422-1.498)	0.478
mt/mt	16 (20)	18 (15)	0.88	(0.409-1.933)	0.766
CYP1A1 m2
wt/wt	37 (46.25)	54 (45)	1.00
wt/mt	28 (35)	49 (40.83)	0.83	(0.446-1.558)	0.569
mt/mt	15 (18.75)	17 (14.17)	1.28	(0.573-2.896)	0.541
CYP1A1 m4
wt/wt	80 (100)	120 (100)	1.00
wt/mt	0 (0)	0 (0)
mt/mt	0 (0)	0 (0)
GSTM1
Present	59 (73.75)	94 (78.34)	1.00
Absent	21 (26.25)	26 (21.66)	1.28	(0.665-2.492)	0.454
GSTT1
Present	51 (63.75)	84 (70)	1.00
Absent	29 (36.25)	36 (30)	1.32	(0.728-2.418)	0.356
GSTM1 & T1
Present	57 (71.25)	102 (85)	1.00
Absent	23 (28.75)	18 (15)	2.28	(1.139-4.599)	0.02^a

^a p < 0.05 Statistically significant.

Figure 1.

Ethidium bromide stained gel showing CYP1A1, GSTM1 & GSTT1 polymorphism. (1.1) CYP1A1m1 genotype analysis by PCR-RFLP method, lane M: 100 bp molecular weight marker, lanes 2, 4, 5 and 7 are wild type alleles; lanes 1 and 3 are heterozygous allele; lane 6 is variant allele.(1.2) CYP1A1 m2 genotype analysis by allele-specific PCR method, lane M: 100 bp molecular weight marker, lanes 1 and 2 represent homozygous wild-type allele (wt/wt) showing amplification with wild-type primer, lanes 3 and 4 represent heterozygous allele (wt/mt) showing amplification with both primer, lanes 5 and 6 represent homozygous mutant type allele(wt/mt) showing amplification with mutant primer. (1.3) CYP1A1 m4 genotype analysis by PCR-RFLP method, Lane M: 50 bp molecular weight marker, lanes 1 to 6 represent homozygous wild type allele. (1.4) PCR products from multiplex amplification of GSTM1 (215 bp), GSTT1 (480 bp) and the internal control, the CYP1A1 gene (312 bp). M = 100 bp DNA marker; lanes: 1, 4 and 6 are GSTM1+/GSTT1+ genotype; lanes 5 and 7 GSTM1+/GSTT1- genotype; lane 3 GSTM1-/GSTT1+ genotypes; lane 2 both GSTM1 and GSTT1 null genotype.

Table 5.

Hypospadias risk associated with CYP1A1, GSTM1 and GSTT1 genotypes adjusted by low birth weight (LBW) and occupational exposure

Genotype	OR^a	95%CI	p-value
CYP1A1 m1
wt/wt	1.00
wt/mt	0.57	(0.287-1.133)	0.109
mt/mt	0.53	(0.223-1.251)	0.147
CYP1A1 m2
wt/wt	1.00
wt/mt	0.69	(0.357-1.343)	0.277
mt/mt	1.11	(0.475-2.577)	0.815
GSTM1
Present	1.00
Absent	0.81	(0.373-1.760)	0.595
GSTT1
Present	1.00
Absent	1.02	(0.532-1.928)	0.968
GSTM1 & T1
Present	1.00
Absent	1.72	(0.823-3.622)	0.149

^a Adjusted for low birth weight and history of occupational exposure.

Discussion

Previously environmental chemicals were generally not considered as a causative factor for hypospadias. Sonawane et al.³² hypothesized that exposure of the developing male fetus to environmental chemicals may be responsible for anomalies of sexual maturation and reproductive function in adult life. In light of the growing number of EDCs reported in human tissue, exposure to environmental contaminants is now being considered in familial clusters because of the high probability of shared exposure.³³ Due to their persistence and stability in the environment, pesticides are common contaminants in ecosystem and are reported to be present in tissues of mothers and children, especially in regions devoted to intensive agriculture.^34,35 Maternal exposure to pesticides has been associated with urogenital malformations, semen quality impairment and testicular, prostate, ovarian and breast cancer.³⁶ Dolk et al. (1998) reported a borderline increase in the incidence of hypospadias among sons of families living near hazardous waste landfill sites in Europe (OR = 1.96, 95% CI = 0.98–3.92).³⁷ In the present study, we also observed that mothers with history of environmental/occupational exposure might have significantly higher risk of giving birth to hypospadiac boys (Table 1).

Several studies have reported that hypospadias is more common among infants with lower birth weight,^38,39 lower placental weight,⁴⁰ lower gestational age⁴¹ and intrauterine growth retardation.^42,43 In the present study, we have observed significantly higher frequency of low birth weight among hypospadias boys indicating an association between low birth weight and elevated risk of hypospadias. One possible explanation is maternal organochlorine pesticide burden and placental transfer of these pesticides to the growing fetus.¹² Longnecker et al. (2002) reported that higher maternal serum levels of p,p’-DDE, a metabolite of DDT that inhibit the binding of androgen to androgen receptors and increased risk of hypospadias.⁴⁴ A Norwegian cohort human milk study (HUMIS) also suggested association of low birth weight with high levels of hexachlorobenzene (HCB) in breast milk.⁴⁵

In those cases where the effects are the most profound, genetic predisposition exacerbated by environmental exposure should be considered. A longitudinal pregnancy study (n = 7928) from the United Kingdom concluded that boys born of vegetarian mothers have an odds ratio of 4.99 for hypospadias (95% CI, 2.1–11.88; p = 0.001).⁴⁶ This study also suggests that sons of vegetarian mothers who consumed only organic products had no hypospadias. Akre et al.⁴⁷ have also reported that pregnancy diet lacking meat and fish appears to increase the risk of hypospadias. This raises the question whether pesticides used on fruits and vegetables are involved in the genesis of hypospadias. Mothers who drank soy milk and consumed soy products delivered a larger proportion of boys with hypospadias, although this was not statistically significant. Our study also reports no significant association between maternal dietary habits (veg /non-veg) and the risk of hypospadias.

We found significantly higher concentration of OCPs in cases than in controls. OCPs may have endocrine-disrupting potential with estrogenic or anti-estrogenic properties. Moreover, β-HCH may be the most toxicologically significant HCH isomer as evidenced earlier for its estrogenic effects in mammalian cells, laboratory animals and fish.⁴⁸ β-HCH with mixture of other OCPs has the ability to generate an estrogenic microenvironment through estrogen receptor (ERα) activation.

γ-HCH also acts as endocrine disruptor and shows both estrogenic and anti-estrogenic effects.⁷ India and Romania are the only countries in the world who still produce γ-HCH and it is estimated that global γ-HCH usage from 1950 to 2000 for agricultural, live stock, forestry, human health and other purpose amounts to around 6,00,000 tons. γ-HCH acts as endocrine disruptor and may be able to react directly or indirectly with hormone structure to alter its function, change the pattern of hormone synthesis or modulate the number of hormone receptors and their affinities for specific molecules. Furthermore, γ-HCH has been found to inhibit cytochrome P450 (CYP) enzymes levels and steroidogenic acute regulatory protein (StAR) expression in cultured rat granulose cells.⁴⁹ StAR protein mediates the intra-mitochondrial transfer of cholesterol to the CYP P450 enzymes and CYP P450 enzymes catalyses steroid hormone biosynthesis.⁵⁰ Impaired levels of sex hormones may interfere with implantation and can induce fetal abnormalities. These observations suggest possible mechanism by which γ-HCH can disrupt endocrine function and may be associated with hypospadias as observed in our study.

In the present study, the prevalence of CYP1A1, GSTM1and GSTT1 gene polymorphism and their association with risk to hypospadias has been investigated in north Indian population. The role of polymorphic genes CYP1A1, GSTM1 and GSTT1 when analyzed as a single genotype has no significant association with hypospadias. However, when both null genotypes GSTM1 and GSTT1 are combined, they suggest an increased risk toward hypospadias. CYP1A1 enzyme metabolizes variety of environmental carcinogens, including PAHs, and produces many reactive intermediates. Several recent investigations have pointed out racial/ ethnic differences in genotype that determine enzymes involved in metabolism/ biotransformation. The CYP1A1 m1/m2 or m1/m2 + m2/m2 genotype has been reported to be associated with significant decreased risk of hypospadias in Japanese population.⁵¹ Contrary to earlier reports, we have not found any significant association between CYP1A1 polymorphism and increased/decreased risk of hypospadias. Moreover, In general, prenatal tissues express low levels of cytochrome P450s compared to adult tissue and the capability to metabolize foreign chemicals is limited.⁵² In the present study, heterozygous CYP1A1m1 (wt/mt) as well as homozygous CYP1A1m1 (mt/mt) were less frequently found in cases than in controls (OR = 0.79, 95% CI = 0.422−1.498; OR = 0.88, 95% CI = 0.409−1.933, respectively). Similar results were also observed with CYP1A1m2 polymorphism, whereas polymorphism in CYP1A1m4 was completely absent.

GSTs are phase II detoxification enzymes that protect cells from toxicants by conjugation with glutathione.²³ The frequency of GSTM1 and GSTT1 deletion individually was slightly higher, though not statistically significant in cases than controls. However, the frequency of simultaneous deletion of both the genes, GSTM1 and GSTT1, were significantly higher in cases (OR = 2.28, 95% CI = 1.139–4.599) than in controls. Our results might suggest that the deletion of both GSTM1 genotype and GSTT1 genotype is associated with increased risk of hypospadias. The inconsistency between our results and other Asian population might be due to ethnic variations. Polymorphism in human xenobiotic metabolizing genes displays parallelism in racial, ethnical and geographic distribution and inter-ethnic differences.^26–28 The Indian population is a major distinct group representing one-sixth of the total world population.⁵³ The north Indian population which is known to be Caucasoid Aryans are ethnically different from South Indians known as Caucasoid Dravidians.⁵⁴ Variant alleles of each mutation are associated with enhanced susceptibility to several diseases.⁵¹ Our recent studies on CYP1A1, GSTM1 and GSTT1 gene deletion in north Indian population reported inter- as well as intra-ethnic variation in comparison to various ethnic population and other Indian population.^30,55 To the best of our knowledge, our study is the first to report an association between hypospadias and genetic polymorphisms of the above-mentioned enzymes in north Indian population.

In conclusion, our results suggests that polymorphism within the CYP1A1 gene may not be associated with the risk of hypospadias, whereas individuals with concomitant deletion of both GSTM1 and GSTT1 genotype may have an impaired ability to eliminate organochlorine pesticides metabolically, thus affecting hormone homeostasis. We found that the occupational exposure history associated with higher risk of hypospadias indicates that maternal aspects may be more relevant. Hence, maternal exposure and higher OCPs levels may affect the risk of hypospadias. Therefore, investigating the association between hypospadias and the maternal exposure to endocrine disrupters and polymorphisms of metabolic enzymes will help in the near future. Further epidemiological studies with larger cross-sectional population need to be performed to clearly elucidate genetic/environmental risk factors for hypospadias.

Footnotes

One of the authors Chandra Shekhar Yadav is grateful to Central Pollution Control Board (CPCB), Ministry of Environment and Forest, Govt. of India, for providing fellowship support. Authors are thankful to Ministry of Environment and Forest, Govt. of India, for providing financial support for the study.

This research work was approved by Institutional Ethical Committee for Human research of University College of Medical Sciences, Delhi, India.

The authors of this manuscript do not have any competing financial or personal interests.

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Polymorphism in CYP1A1,GSTMI,GSTT1 genes and organochlorine pesticides in the etiology of hypospadias

Abstract

Keywords

Introduction

Materials and methods

Study population

Extraction, cleanup and quantification of OCPs from blood samples

Genotyping

CYP1A1 genotyping

GSTM1 and GSTT1 genotyping

Statistical analysis

Results

Discussion

Footnotes

References