Published in Volume
120, Issue 3
(March 1, 2010)J Clin Invest.
Copyright © 2010, American Society for Clinical Investigation
Urolithiasis and hepatotoxicity are linked to the anion transporter Sat1 in mice
1School of Biomedical Sciences, University of Queensland, Brisbane, Australia.
2Department of Medical Genetics and the Child and Family Research Institute, University Of British Columbia, Vancouver, Canada.
3Pathology Department, Mater Misercordiae Hospital, South Brisbane, Australia.
Address correspondence to: Daniel Markovich, School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia. Phone: 61.7.3365.1400; Fax: 61.7.3365.1766; E-mail:
First published February 15, 2010
Submitted: August 18,
2009; Accepted: January 6,
Urolithiasis, a condition in which stones are present in the urinary system, including the kidneys and bladder, is a poorly understood yet common disorder worldwide that leads to significant health care costs, morbidity, and work loss. Acetaminophen-induced liver damage is a major cause of death in patients with acute liver failure. Kidney and urinary stones and liver toxicity are disturbances linked to alterations in oxalate and sulfate homeostasis, respectively. The sulfate anion transporter–1 (Sat1; also known as Slc26a1) mediates epithelial transport of oxalate and sulfate, and its localization in the kidney, liver, and intestine suggests that it may play a role in oxalate and sulfate homeostasis. To determine the physiological roles of Sat1, we created Sat1–/– mice by gene disruption. These mice exhibited hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate stones in their renal tubules and bladder. Sat1–/– mice also displayed hypersulfaturia, hyposulfatemia, and enhanced acetaminophen-induced liver toxicity. These data suggest that Sat1 regulates both oxalate and sulfate homeostasis and may be critical to the development of calcium oxalate urolithiasis and hepatotoxicity.
Oxalate is a metabolic end product excreted into the urine (1), whereas sulfate (SO42–) is highly reabsorbed by the kidneys (2, 3), playing important roles in many biochemical reactions. The regulation of oxalate and sulfate homeostasis is complex, with the kidney and liver playing critical roles. The sulfate anion transporter–1 (Sat1; ref. 4) is localized to the basolateral membrane of the renal proximal tubule (5) and the sinusoidal membrane of hepatocytes (6), where it mediates anion exchange (2, 7, 8). The human SAT1 (9) and mouse Sat1 (7) genes — also known as SLC26A1 and Slc26a1, respectively — are located within exon 2/intron 2 (on the opposite strand) of the α-L-iduronidase (IDUA) gene (10). IDUA deficiency leads to mucopolysaccharidosis type I, a lysosomal storage disorder resulting from defective lysosomal degradation of glycosaminoglycans (11). Despite numerous studies characterizing Sat1 transport properties and tissue localization (2, 9, 12, 13), the physiological roles of Sat1 in vivo have not been resolved. To our knowledge, Sat1 has not been linked to any human disorders, and its role within the IDUA gene is unknown; therefore, we generated a Sat1–/– mouse by targeted disruption in order to characterize its functions in vivo. Sat1–/– mice showed perturbations in oxalate and sulfate homeostasis, leading to urolithiasis and hepatotoxicity, respectively.
Our targeting strategy replaced Sat1 exon 3 with a neomycin resistance (neoR) cassette (Figure 1A). Genomic Southern and PCR analyses confirmed deletion of Sat1 in DNA from Sat1–/– mice (Figure 1, B–D). Genotypes of 303 mice were determined by PCR (Figure 1E): 86 were Sat1+/+ (approximately 28%), 146 were Sat1+/– (approximately 48%), and 71 were Sat1–/– (approximately 24%), close to the Mendelian ratio of 1:2:1. This finding indicates that loss of Sat1 is not embryonic lethal. Because Sat1 is localized within Idua (10), our targeting strategy was designed to selectively disrupt Sat1 without affecting Idua (Figure 1F). We confirmed this by determining that RT-PCR (using primers P6 and P7; Figure 1F and Supplemental Table 1; supplemental data available online with this article; doi:
10.1172/JCI31474DS1) amplified similar amounts of Idua mRNA from kidney, liver, and ileum of Sat1–/– and Sat1+/+ mice (data not shown). Idua enzyme activity in Sat1–/– liver (0.029 ± 0.003 nM/min/mg protein; n = 3) and kidney (0.015 ± 0.001 nM/min/mg protein; n = 3) were similar to that in Sat1+/+ liver (0.025 to 0.050 nM/min/mg protein) and kidney (0.010 to 0.032 nM/min/mg protein), respectively (14), which suggests that loss of Sat1 does not alter Idua function. No differences in body weight, tail length, or obvious distinguishing body features were observed in male and female Sat1–/– mice compared with Sat1+/– or Sat1+/+ mice (n = 15–40 per group) from 1 to 24 weeks of age (data not shown). Gross histological analyses of the liver and brain, where Sat1 is normally expressed, showed no structural differences between Sat1–/– and Sat1+/+ mice (data not shown).
Targeted disruption of Sat1.
(A) Sat1 targeting strategy. Exons (boxes 1–4), restriction sites, probes, and primers P1–P5 are shown. NeoR, neomycin resistance sequence; TK, thymidine kinase sequence. (B) Southern analysis of Hind III– and Not I–digested DNA from Sat1+/+, Sat1+/–, and Sat1–/– mice. Probe A detected 9.9-kb wild-type and 5.2-kb targeted allele fragments. (C) Southern analysis of Bcl I–digested DNA from Sat1+/+, Sat1+/–, and Sat1–/– mice. The neoR probe detected a single 4.7-kb fragment in Sat1+/– and Sat1–/– mice. (D) Forward (P4) and reverse (P5) primers amplified an 8.9-kb product in DNA samples from Sat1+/– and Sat1–/– mice. M, molecular mass ladder; Neg, negative control. (E) Primers P1 and P2 amplified a 0.5-kb wild-type fragment; P1 and P3 amplified a 0.4-kb targeted allele fragment. (F) Selective disruption of the Sat1 gene located on the opposite strand within the Idua gene. Sat1 exons (white boxes 1–4), Idua exons (black boxes 1–14), primers P6 and P7, and neomycin resistance sequence are shown.
A single 3.8-kb Sat1 transcript was detected in liver and kidney, but not ileum, of Sat1+/+ and Sat1+/– mice, but not Sat1–/– mice (Figure 2B). Truncated cDNAs were amplified from Sat1–/– and Sat1+/– kidney mRNA (Figure 2C). Sequence analysis revealed Sat1 exons skipping from 2 to 4 (Figure 2A), with this cDNA lacking the first 587 bases of the Sat1 coding region, including the ATG start codon. Sat1 transcripts were amplified in the distal ileum, cecum, and proximal colon (Figure 2D). Sat1 protein was detected in basolateral membrane vesicles (BLMVs) from distal ileum, cecum, proximal colon (Figure 2E), and renal cortex (data not shown) of Sat1+/+ mice, but not Sat1–/– mice. Oxalate and SO42– transport was strongly reduced in BLMVs isolated from kidney, liver, distal ileum, cecum, and proximal colon of Sat1–/– mice compared with Sat1+/+ mice (Table 1), with no differences observed in brush border membrane vesicles (BBMVs) of Sat1+/+ and Sat1–/– mice (data not shown).
Analysis of Sat1 protein and mRNA. (A) Schematic of Sat1 cDNA and the predicted knockout cDNA lacking exon 3. Exons 1–4 (boxes), protein coding sequences (shading) of wild-type (3,821 nucleotides) and Sat1 knockout (3,201 nucleotides) cDNA, and primers P8 and P9 are shown. (B) Northern analysis of RNA from liver (L), kidney (K), and ileum (I) of Sat1+/+, Sat1+/–, and Sat1–/– mice. RNA was hybridized with a 32P-labeled Sat1 cDNA (top), showing the 3.8-kb transcript, and GAPDH cDNA (bottom). (C) Total RNA from kidney was RT-PCR amplified. A 1,716-bp wild-type product and a 1,096-bp knockout Sat1 cDNA product (top) were amplified using primers P8 and P9. A 983-bp product (bottom) was amplified using GAPDH primers. (D) Total RNA from Sat1+/+ intestinal segments was RT-PCR amplified. A 1,716-bp product was amplified using primers P8 and P9. A 983-bp product (bottom) was amplified using GAPDH primers. (E) Western analysis of protein from intestinal BLMVs and apical BBMVs. The Sat1 antibody detected a 75.4-kDa protein in Sat1+/+ BLMVs, but not in Sat1+/+ BBMVs or in Sat1–/– mice.
BLMV transport of oxalate and sulfate
Role of Sat1 in the urinary system. Sat1–/– mice had infiltration of leukocytes around renal cortical vessels that was absent in Sat1+/+ mice (Figure 3, A and B). Calcium oxalate crystals were observed in the lumen of kidney cortical tubules and the bladder of Sat1–/– mice, but not Sat1+/+ mice (Figure 3, C–F). Crystals in the cortex and outer medullary regions of the Sat1–/– kidney (Figure 4, A and B) were mainly composed of calcium, oxygen, and carbon (Figure 4C), indicating primary composition of calcium oxalate.
Urolithiasis in Sat1–/– mice.
(A and B) Representative H&E-stained kidney sections showing infiltration of leukocytes (arrow) around the renal cortical vessels in 100% of Sat1–/– mice (n = 8), absent in Sat1+/+ mice. (C–F) Representative Yasue-stained kidney (C and D) and bladder (E and F) sections showing calcium oxalate stones (dark staining) in kidney tubules of 47% Sat1–/– mice (n = 15) and bladders of 26% Sat1–/– mice (n = 19), but not in any Sat1+/+ mice (n = 10). Gross histological analyses of additional tissues in which Sat1 is expressed (liver and brain) showed no structural differences between Sat1–/– and Sat1+/+ mice (not shown). Scale bars: 100 μm (A and B); 500 μm (C and D); 2 mm (E and F).
Energy dispersive X-ray spectrometry analysis of kidney stones in Sat1–/– mice.
(A and B) Representative electron micrograph (A) and energy dispersive X-ray micrograph (B) showing kidney stones (arrowheads in B) in the renal cortex and outer medullary region of Sat1–/– mice. Higher-magnification view of the boxed region in B is shown in the inset. Scale bar: 1 mm; 100 μm (inset). (C) Elemental profile of kidney stones showing abundance of calcium (11.01% ± 0.03%), oxygen (36.53% ± 0.22%), and carbon (51.46% ± 0.08%), as well as trace levels (≤0.30%) of sodium, magnesium, aluminium, sulfur, chromium, and iron. CPS, counts per second; keV, kiloelectron volts.
Role of Sat1 in the liver. Because sulfate is involved in detoxifying xenobiotics such as acetaminophen (APAP), and APAP administration causes rapid plasma sulfate depletion in humans and rodents (15, 16), we tested whether hepatic phase II sulfonation was affected in Sat1–/– mice. As shown previously in our Nas1–/– mice (17), 12-hour administration of 250 mg/kg APAP led to a 4-fold increase in serum alanine aminotransferase (ALT) in Sat1–/– mice compared with Sat1+/+ mice (Figure 5A), and extensive liver necrosis was observed in Sat1–/– mice, but not Sat1+/+ mice (Figure 5, C and D, and Supplemental Table 2). A single 250-mg/kg dose of APAP led to significantly greater reductions in glutathione (GSH) levels in Sat1–/– mice (reduced by approximately 96%) than in Sat1+/+ mice (reduced by approximately 80%) after 2 hours; after 12 hours, basal GSH levels were restored in both Sat1–/– and Sat1+/+ mice (Figure 5B).
Increased hepatotoxicity in Sat1–/– mice.
(A and B) Serum ALT and liver GSH levels in Sat1+/+ (white bars) and Sat1–/– (black bars) mice 0, 2, and 12 hours after administration of 250 mg/kg APAP. Results are mean ± SEM (n = 4–8 per group). *P < 0.05 versus time 0 Sat1+/+; #P < 0.05 versus time 0 Sat1–/–. (C and D) Representative H&E-stained liver sections of Sat1+/+ and Sat1–/– mice (n = 6–7 per group) 12 hours after administration of 250 mg/kg APAP, showing liver necrosis in Sat1–/– mice. Scale bars: 100 μm.
Role of Sat1 in sulfate and oxalate homeostasis. Blood and urine profiling of Sat1–/– and Sat1+/+ mice revealed that plasma oxalate levels increased by approximately 60%, whereas serum SO42– levels were reduced by approximately 60%, in Sat1–/– mice compared with Sat1+/+ mice (Table 2). Urine oxalate/creatinine and SO42–/creatinine ratios and fractional excretion index (FEI) for SO42– were significantly increased in Sat1–/– mice. Urinary glycolate/creatinine levels were not altered in Sat1–/– or Sat1+/+ mice. A decrease in cecal oxalate and an increase in cecal sulfate content was observed in Sat1–/– mice.
Blood, urinary, and cecal profiles of Sat1+/+ and Sat1–/– mice
Overlapping genes in eukaryotes have been thought to be rare, but more recent analyses of the human and mouse genomes (18) suggest that overlapping transcripts in mammals may be more common than previously thought. Early studies proposed that Sat1 localization within the Idua gene may produce an antisense effect between the Sat1 and Idua transcripts and act as a potential mechanism for coordinating expression of these genes (10). Selective disruption of Idua in mice showed no change in Sat1 mRNA levels (14), and here we observed no change in the functional activity of Idua in tissues of Sat1–/– mice, which suggests that antisense effects are unlikely to play a significant role in regulating the expression of Sat1 and Idua genes.
Urolithiasis is the most common chronic disorder of the urinary tract, with 70%–80% of stones being calcium oxalate crystals (19). Our observation of calcium oxalate crystals in the bladder and renal cortical and medullary tubules of Sat1–/– mice suggests that Sat1 plays a major role in renal oxalate excretion. Infiltration of leukocytes around renal cortical vessels of Sat1–/– mice suggests ureteral obstruction or obstructive uropathy (20). Sat1–/– mice had significantly elevated plasma oxalate levels, a greater than 2-fold increase in urine oxalate/creatinine ratio, no changes in urinary glycolate/creatinine levels (an indicator of hepatic oxalate production), and reduced oxalate transport in BLMVs of distal intestinal segments, which suggests that the hyperoxaluria in Sat1–/– mice arises most likely from hyperoxalemia rather than altered renal tubular oxalate handling. No changes were observed for whole blood pH; urine pH; urinary protein levels; cecal water content; serum levels of creatinine, Cl–, PO42–, Na+, K+, and Ca2+; and FEIs of PO42– and Ca2+ between Sat1–/– and Sat1+/+ mice (Table 2), which suggests that homeostasis of these ions was not perturbed in Sat1–/– mice. Sat1–/– mice had significantly decreased serum sulfate levels, increased urine sulfate/creatinine, and increased FEI sulfate, suggesting that the hyposulfatemia in Sat1–/– mice most likely arises from increased renal excretion, as previously observed in Nas1–/– mice (21). This agrees with the present observation of strongly reduced sulfate transport across renal and liver BLMVs of Sat1–/– mice, which suggests that Sat1 is the predominant sulfate transporter on the renal proximal tubular basolateral and liver sinusoidal membranes. The hyposulfatemia and FEI sulfate values in Sat1–/– mice were comparable to those in Nas1–/– mice (21), which suggests that Sat1, together with NaS1, plays a crucial role in renal sulfate reabsorption and maintenance of serum sulfate levels. Despite Sat1 being expressed in several other tissues, including heart, brain, calvaria, and skeletal muscle (7), its role in these tissues — and to what degree these tissues contribute to circulating sulfate levels — is yet unknown. Our identification of Sat1 mRNA in the distal ileum, cecum, and proximal colon and Sat1 protein on the basolateral membrane of these intestinal segments suggests that Sat1 plays a role in distal intestinal oxalate and sulfate transport. This is corroborated with reduced oxalate transport in BLMVs from distal ileum, cecum, and proximal colon as well as decreased cecal oxalate content, indicating that the hyperoxalemia in Sat1–/– mice is most likely caused by reduced intestinal secretion of oxalate, as previously observed in Slc26a6–/– mice (22, 23). Our data suggest that Sat1 is the basolateral anion exchanger, which, together with Slc26a6 on the brush border membrane (22, 23), facilitates intestinal oxalate secretion.
Sulfate is required for detoxification of xenobiotics, including APAP (3), the leading cause of drug-related liver failure in humans (3, 24). Sat1–/– mice had increased APAP-induced liver toxicity, which suggests that the hyposulfatemia in Sat1–/– mice limits the supply of sulfate for APAP metabolism, as previously demonstrated in Nas1–/– mice (17). High doses of APAP cause rapid depletion of GSH, saturation of the sulfonation and glucuronidation pathways, and increased production and half-life of the toxic reactive intermediate N-acetyl-p-benzoquinone, which leads to cellular damage and death (25). Depletion of hepatic GSH levels and the rate of cellular GSH repletion are critical factors in APAP hepatotoxicity (26, 27). This agrees with our observations of decreased hepatic GSH levels and increased APAP-induced hepatotoxicity in Sat1–/– mice. The loss of Sat1 from the sinusoidal membranes of hepatocytes, where it facilitates sulfate import for sulfonation reactions (6), as well as the hyposulfatemia, exacerbate hepatic sulfate depletion and drug-induced liver damage by drugs such as APAP. The roles of Sat1 polymorphisms (28) in drug metabolism rates are currently unknown, but the present study suggests that Sat1 may play an important role.
In conclusion, this is the first study to our knowledge to demonstrate Sat1 localization on the basolateral membrane of intestinal cells and show that loss of Sat1 expression leads to urinary stone formation and drug-induced hepatotoxicity, owing to altered homeostasis of oxalate and sulfate, respectively. Disturbed oxalate homeostasis led to hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate urolithiasis, whereas perturbed sulfate homeostasis led to hyposulfatemia, hypersulfaturia, and enhanced hepatotoxicity in Sat1–/– mice. The present study provides an animal model for characterizing the etiology and mechanisms underlying calcium oxalate urolithiasis and the hepatic phase II sulfonation pathway and represents a therapeutic target for investigating human kidney stone formation and drug-induced hepatotoxicity in the general population.
Targeting vector construction. Fragments of Sat1 were PCR amplified and used to create the targeting vector shown in Figure 1A. A 2.3-kb fragment containing exons 1 and 2, and an 8.0-kb fragment containing exon 4, were subcloned on either side of the phosphoglycerate kinase–neoR expression cassette of the pCAG Neo vector. Upon homologous recombination, this vector deleted approximately 0.9 kb of Sat1 genomic sequences, including the ATG start codon in exon 3 (Figure 2A).
Generation and identification of Sat1–/– mice.
The targeting construct was electroporated into 129JSv mouse ES cells. Transfectants were selected for resistance to neomycin and ganciclovir (14). Individual clones that had undergone homologous recombination were identified by Southern blot analysis using an external 1.3-kb Pst I/Ava I fragment upstream of exon 1 as a probe (Figure 1A). Probe A detected a 9.9-kb Hind III wild-type allele fragment and a 5.2-kb Hind III/Not I recombinant allele fragment. Positive ES cell clones were injected into CD1 blastocysts that were implanted into foster mothers. Chimeric mice were bred with CD1 mice, and heterozygous offspring were intercrossed to produce Sat1–/– mice. Tail biopsies were further analyzed by Southern blotting using a neoR probe (Figure 1A) and by using PCR to amplify an 8.9-kb recombinant allele fragment. PCR was performed using primer P4 in the neoR gene cassette and antisense primer P5 downstream of exon 4 (Supplemental Table 1 and Figure 1A). Studies were performed on male mice with a mixed genetic background (129JSv and CD1). Mice were maintained on a 12-hour light/dark cycle and fed ad libitum on standard rodent chow (diet no. AIN93G; Glen Forrest Stockfeeders). All animal experiments were approved by the University of Queensland Anatomical Biosciences Ethics Committee.
Genotyping of mice. Mice were genotyped using primer P1 in intron 2 and antisense primer P3 in the neoR gene cassette to PCR amplify a 0.4-kb fragment of the recombinant Sat1 allele (Supplemental Table 1 and Figure 1A). Primer P1 and antisense primer P2 in exon 3 were used to amplify a 0.5-kb fragment of the wild-type Sat1 allele (Supplemental Table 1 and Figure 1A). Cycle parameters were as follows: 95°C for 10 seconds followed by 35 cycles of 95°C for 10 seconds, 58°C for 30 seconds, and 68°C for 3 minutes.
Analysis of RNA. Total RNA (10 μg) was separated on a 1.2% agarose formaldehyde gel in MOPS buffer and transferred to Hybond-XL nylon membranes (Amersham). The blots were probed with a 1.4-kb Sat1 cDNA and a 0.6-kb mouse GAPDH cDNA probe. For RT-PCR, total RNA (2 μg) was reverse transcribed using random hexamers and Moloney Murine Leukemia Virus reverse transcriptase (Progen), as recommended by the manufacturer. Primer P8 in exon 2 and antisense primer P9 in exon 4 (Figure 2A and Supplemental Table 1) were used to amplify Sat1 cDNA fragments. GAPDH cDNAs were RT-PCR amplified using established primer sequences (17).
Membrane vesicle preparation, transport studies, and Western blotting. BLMVs and BBMVs were isolated from mouse kidney, liver, and intestine using previously described methods (29, 30). Vesicles were preincubated in buffer containing 120 mM potassium gluconate, 30 mM KCl, and 15 mM HEPES pH 8.2 for 2 hours at 20°C. The uptakes of SO42– (0.1 mM) and oxalate (20 μM), each performed in quadruplicate, were measured at 10 seconds (SO42–) and 30 seconds (oxalate) in media containing 150 mM potassium gluconate and 15 mM HEPES, pH 8.2, by the rapid filtration technique (23, 31). BLMV protein (5 μg) was separated on a 10% SDS polyacrylamide gel and transferred to nylon membrane (Immobilon-FL membrane; Millipore). Sat1 proteins were detected with a Sat1 antibody (5) provided by L.P. Karniski (University of Iowa, Iowa City), with IRDye CW800-labeled anti-mouse IgG (diluted 1:20,000) and the Odyssey Imaging System (LI-COR Biotechnology). All experiments were repeated at least twice.
Biochemical analyses. Mouse blood was collected from the submandibular vein using an animal bleeding lancet (MEDIpoint Inc.). Single urine samples were collected upon spontaneous micturition during mouse handling, prior to blood collection. A turbidimetric assay was used to measure free sulfate levels (17). Oxalate was measured by ion chromatography with suppressed conductivity detection using a Dionex ICS2000 running Chromeleon 6.80 (32, 33). Upon collection of 100 μl serum and urine, 10 μl HCl (8M) was quickly added, and the samples were frozen at –80°C to prevent oxalate precipitation and oxalogenesis. Serum and urine creatinine levels were quantitated enzymatically using a Vitros 5.1 FS chemistry analyzer (Ortho-Clinical Diagnostics). Serum PO42–, Na+, K+, Cl–, Ca2+, and ALT concentrations were assayed by a commercial pathology laboratory (Sullivan Nicolaides Pathology) using a Vitros 250 chemistry analyzer (Ortho-Clinical Diagnostics). Urinary glycolate levels were quantitated using an Agilent 6890 GC and 5973A MSD running MSD Chemstation B.01.00. Urine total protein levels were quantitated using a protein microassay kit (BioRad). Whole blood and urine pH was measured with an ISFET pH meter (Shindengen). Hepatic GSH levels were assayed using a commercially available kit (Cayman Chemical Co.). H2O content of cecal samples was determined by weighing samples before and after they were desiccated under vacuum for 30 hours at 25°C. Kidney and liver IDUA activity was measured using a fluorometric assay (34).
Histopathological analysis. Tissues were dissected into approximately 50 volumes of 10% buffered formalin and fixed for 3 days prior to paraffin embedding. Embedded tissue was sectioned, stained with H&E or by the Yasue histochemical method (35) for detecting calcium oxalate crystals, and examined by light microscopy. Liver sections were scored blinded to genotype for the severity of degeneration and necrosis using a 0–5 scale as previously described (36).
APAP administration. APAP (Sigma-Aldrich) was dissolved in saline and filter-sterilized. At approximately 3 months of age, mice received i.p. injection of a 250-mg/kg dose of APAP (n = 4–8 per group). Mice were sacrificed at 2 or 12 hours after APAP administration.
Statistics. Statistical significance between Sat1+/+ and Sat1–/– or Sat1+/– groups was assessed using an unpaired Student’s 2-tailed t test. Statistical significance between Sat1+/+ and Sat1–/– groups with respective controls at time point 0 was evaluated using 1-way ANOVA followed by Duncan’s multiple range test. Differences in histology scores were analyzed by the Mann-Whitney test. In all cases, a P value less than 0.05 was considered significant.
We thank S. Gibson, K. Colobong, P. Addison, I. Wilkie, and I. Shiels for scientific assistance. L.A. Clarke holds an investigatorship award through the Child and Family Research Institute. This work was supported in part by the Australian Research Council and the National Health and Medical Research Council.
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