Total oxidant/antioxidant status in sera of patients with thyroid cancers

in Endocrine-Related Cancer

Oxidative stress is considered to be involved in the pathophysiology of all cancers. In order to evaluate the total oxidant/antioxidant status in patients with thyroid cancer and to investigate the relationship between oxidative stress parameters and serum thyroid profiles among thyroid cancer patients and various controls, we determined oxidative status including total antioxidant status (TAS) and total oxidant status (TOS) and calculation of oxidative stress index (OSI) in sera in 82 thyroid cancer patients, 56 benign thyroid disease patients, and 50 healthy controls. It was found that serum TAS levels were significantly lower in patients with thyroid cancer than in controls (P<0.001), while serum TOS levels and OSI values were significantly higher (both P<0.001) in the cancer patients. No significant correlations were observed between various oxidative stress markers and thyroid profiles in either the thyroid cancer patients or the controls. Receiver operating characteristic curve analysis demonstrated that OSI was the best indicator for distinguishing cancer patients from benign thyroid diseased or healthy controls, followed by TOS and TAS. Risk estimate statistics also indicated that TOS and/or OSI were good risk factors to discriminate patients with thyroid cancer from two controls. These findings suggested that oxidants are increased and antioxidants are decreased in patients with thyroid cancer. OSI may be a more useful oxidative stress biomarker than TAS and TOS for monitoring the clinical status of thyroid cancer patients.

Abstract

Oxidative stress is considered to be involved in the pathophysiology of all cancers. In order to evaluate the total oxidant/antioxidant status in patients with thyroid cancer and to investigate the relationship between oxidative stress parameters and serum thyroid profiles among thyroid cancer patients and various controls, we determined oxidative status including total antioxidant status (TAS) and total oxidant status (TOS) and calculation of oxidative stress index (OSI) in sera in 82 thyroid cancer patients, 56 benign thyroid disease patients, and 50 healthy controls. It was found that serum TAS levels were significantly lower in patients with thyroid cancer than in controls (P<0.001), while serum TOS levels and OSI values were significantly higher (both P<0.001) in the cancer patients. No significant correlations were observed between various oxidative stress markers and thyroid profiles in either the thyroid cancer patients or the controls. Receiver operating characteristic curve analysis demonstrated that OSI was the best indicator for distinguishing cancer patients from benign thyroid diseased or healthy controls, followed by TOS and TAS. Risk estimate statistics also indicated that TOS and/or OSI were good risk factors to discriminate patients with thyroid cancer from two controls. These findings suggested that oxidants are increased and antioxidants are decreased in patients with thyroid cancer. OSI may be a more useful oxidative stress biomarker than TAS and TOS for monitoring the clinical status of thyroid cancer patients.

Keywords:

Introduction

Thyroid cancer is the most common endocrine malignancy and its incidence continues to rise yearly. Thyroid carcinoma, in most cases, presents clinically as a solitary nodule or as a dominant nodule within a multinodular thyroid gland. The challenge to clinicians is to discriminate between the minority of thyroid nodules (5–15%) that harbor malignancies and the majority of cases that can be managed conservatively (Boelaert 2009). Thus, it is very important to identify one or more markers to distinguish thyroid cancer from different types of thyroid disease.

Cellular oxidative damage is a well-established general mechanism of cell and tissue injury that is primarily caused by free radicals and reactive oxygen species (ROS). Low levels of ROS are indispensable in many biochemical processes (Valko et al. 2007); however, overproduction and/or inadequate removal of ROS can result in oxidative stress, which is characterized as an imbalance between the formation of active oxygen metabolites and the rate at which they are scavenged by enzymatic and non-enzymatic antioxidants. Oxidative stress can participate in the pathogenesis and complications of many diseases (including cancer; Uttara et al. 2009).

Thyroid hormones regulate oxidative metabolism and thus play an important role in free radical production (Erdamar et al. 2008). Thyroid hormones regulate the synthesis and degradation of enzymes, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase, and non-enzymatic antioxidants, such as vitamin E and C, glutathione, uric acid, ferritin, transferrin, and ceruloplasmin. Undoubtedly, the changes in these enzymes and non-enzymatic substances affect the redox balance in the body and, in turn, enzymatic feedback regulates thyroid function. One of the major effects of thyroid hormones is to increase mitochondrial respiration, which results in upregulation of ROS, leading to oxidative damage to membrane lipids (Erdamar et al. 2008).

In recent years, interest has grown in studying the role played by oxidative stress in thyroid carcinogenesis by investigating one or more of the antioxidant markers, including SOD (Laatikainen et al. 2010, Lassoued et al. 2010, Erdamar et al. 2010), catalase (Lassoued et al. 2010), GPx (Lassoued et al. 2010, Erdamar et al. 2010), vitamin E (Alkhenizan and Hafez 2007), vitamin C (Liu et al. 2010), glutathione (Du et al. 2009), and/or uric acid (Abiaka et al. 2001). However, the measurement of different antioxidant molecules separately was not only impractical but also held no clinical significance. Because the effects of antioxidants can be additive and measuring individual antioxidants separately is time consuming and labor intensive, a measurement of the combined activities of all antioxidants or the total antioxidant status (TAS) is often used to estimate the overall antioxidative status (Erel 2004). Likewise, total oxidant status (TOS) is measured to determine a patient's overall oxidation state (Erel 2005). Furthermore, the oxidative stress index (OSI), which is calculated as the ratio of TOS to TAS, may be a more accurate index of oxidative stress in the body because it is a comprehensive measurement of TAS and TOS.

Oxidative stress is common in the thyroid tissue during utilization of H2O2 for thyroxine (T4) synthesis, the inflammation produces ROS, and when tumor has active proliferation (Akinci et al. 2008). Therefore, oxidative stress has been reported to be consistently associated with hyperthyroidism or hypothyroidism (Erdamar et al. 2008). However, to date, our understanding of the roles of the oxidative/antioxidative systems in the carcinogenesis of thyroid cancer and their relationships to thyroid profiles is limited. To address this, we evaluated the levels of TAS, TOS, and OSI in thyroid cancer patients, benign thyroid disease patients, and healthy controls and analyzed the relationship among these oxidative stress parameters, thyroid profiles, and thyroid cancer.

Materials and methods

Subjects

From March 2005 to December 2010, 82 patients with thyroid cancer (35 males and 47 females) were involved in this study. All patients were newly diagnosed as having thyroid cancer based on clinical laboratory investigations confirmed by fine needle aspiration. The mean age of the patients was 33.4±7.9 years with a range of 12–53 years. Of these 82 cancer patients (cancer group), there were 53 papillary thyroid cancer (PTC) patients, 18 follicular thyroid cancer patients, six anaplastic differentiated thyroid cancer (ATC) patients, and five medullary thyroid cancer patients. In addition, there were 56 benign thyroid disease patients (22 males and 34 females; age range 15–62 years) that served as disease controls, including 36 patients with hyperthyroidism due to Graves' disease (hyperthyroidism group) and 20 patients with hypothyroidism due to Hashimoto's thyroiditis (hypothyroidism group). These patients were also newly diagnosed cases of clinical dysthyroidism.

Age-matched subjects from the same region (n=50; 19 males and 31 females; age range 17–56 years) were used as healthy controls (healthy group (HG)). HG blood samples were measured in parallel to obtain normal base line values.

The study protocol was approved by the Medical Ethics Committee of Mianyang Central Hospital, Sichuan province, China, and written informed consent was obtained from all legal guardians of tested participants.

Blood samples

After overnight fasting, blood was drawn by venipuncture at the antecubital vein from the subjects between 0730 and 1000 h. Approximately 5 ml blood was collected into a BD Vacutainer SST II ADVANCE tube (Becton Dickinson, Rutherford, NJ, USA) for analysis of oxidative stress markers and thyroid profiles. After 1 h, blood samples were centrifuged at 700 g for 15 min, and sera samples were collected and stored at −30 °C until analysis within 48 h.

Thyroid profiles

Thyroid profiles were assessed by estimation of serum-free triiodothyronine (FT3), free T4 (FT4), and thyrotropic-stimulating hormone (TSH), which were assayed by a chemiluminescent assay method using a LIAISON analyzer with original kits obtained from DiaSorin (DiaSorin S.p.A, Saluggia, Italy). The FT3 to FT4 ratio (FT3/FT4) was also calculated.

TAS determination

TAS was measured colorimetrically using the TAS kit (Eurobiomed Randox, London, UK). This assay relies on the ability of antioxidants in the sample to inhibit the formation of ABTS+ from oxidation of ABTS (2.2′-azino-di-(3-ethylbenz-thiazoline sulfonate)) by metmyoglobin (a peroxidase). An antioxidant of known concentration (1.65 mmol/l) was used as a standard for the calculation of antioxidant levels in the samples. Values of TAS were expressed as mmol Trolox equivalent/l (mmol Trolox equiv./l).

TOS determination

Serum TOS was measured using Erel's TOS method (Harma et al. 2005), which is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidative species in acidic medium and the measurement of the ferric ion by xylenol orange. The test parameters were as follows: end-point measurement, serum 10 μl, R1 200 μl, R2 50 μl, 10 min at 37 °C, read points 34, primary wavelength 560 nm, and secondary wavelength 800 nm. The results were expressed in μmol H2O2 equivalent/l (μmol H2O2 equiv./l).

Oxidative stress index

The TOS to TAS ratio was regarded as the OSI (Aycicek et al. 2005, Harma et al. 2005) and was calculated as follows: OSI (arbitrary unit)=((TOS, μmol H2O2 equiv./l)/(TAS, μmol Trolox equiv./l)×100) (Aycicek & Erel 2007).

Statistical analysis

All data were expressed as the mean±s.d. and/or range (minimum–maximum). One-way ANOVA was used to determine the significance of biochemical parameters among the study groups. The least significant difference test was used for multiple comparisons to estimate the difference between the two study groups. Multiple linear regression analysis was performed to evaluate the associations between serum levels of oxidative stress markers and thyroid profiles. Receiver operating characteristic (ROC) curves, depicting the ability to discriminate between controls and malignant conditions, were plotted for the oxidative stress markers and reported the area under the curve (AUC) and its 95% confidence interval (CI). P values <0.05 were considered statistically significant. All statistical analyses were carried out using the statistical package program version SSPS 17.0 (SPSS, Inc., Chicago, IL, USA).

Results

In this study, hyperthyroidism was defined as a basal serum TSH <0.4 mIU/l, FT4 >1.9 ng/ml, and FT3 >4.2 pg/ml. Hypothyroidsm was defined as basal serum TSH >4.0 mIU/l, FT4 <0.8 ng/ml, and FT3 <1.8 pg/ml. Healthy controls included demonstrated a basal serum TSH 0.4–3.8 mIU/l, FT4 0.8–1.7 ng/ml, and FT3 2.2–4.2 pg/ml.

The results are expressed as mean±s.d. with minimum and maximum values shown in Table 1. Samples of various concentrations were assayed to estimate repeatability and reproducibility of each assay. The intra-assay and inter-assay coefficients of variation (CVi, CVd) are presented in Table 2.

Table 1

Serum levels of oxidative stress markers and thyroid profiles in study groups (above: mean±s.d., below: min.–max.)

ItemsCG (82)HpeG (36)HpoG (20)HG (50)Statistic values
TAS1.37±0.20*,‡,§1.52±0.131.53±0.111.67±0.15F=32.951
1.00–1.701.33–1.771.32–1.691.45–1.95P=0.000
TOS21.34±2.82*,‡,§15.79±1.4116.08±1.4714.87±1.01F=129.850
16.23–26.5513.51–18.8213.35–18.0813.22–16.95P=0.000
OSI1.60±0.31*,‡,§1.05±0.111.06±0.110.89±0.07F=129.069
1.01–2.330.82–1.230.91–1.280.69–1.09P=0.000
TSH0.221±0.354*0.028±0.027*20.665±4.054*1.627±0.766F=1269.993
0.002–2.0750.004–0.08815.908–29.6640.677–3.050P=0.000
FT32.82±0.59*7.16±1.40*1.42±0.37*3.02±0.42F=351.354
1.52–4.195.45–9.700.95–1.952.25–3.95P=0.000
FT41.10±0.3115.76±6.62*0.53±0.161.22±0.24F=249.572
0.53–1.695.62–28.120.30–0.820.82–1.67P=0.000
FT3/FT42.80±1.050.57±0.32*2.98±1.392.56±0.53F=60.484
0.90–5.460.21–1.501.45–6.231.42–3.80P=0.000

Comparison with HG, *P<0.001, P<0.05; comparison with HpeG, P<0.001; comparison with HpoG, §P<0.001. Least significant difference (LSD) test. TAS, TOS, and OSI units are expressed as mmol Trolox equivalent/l, μmol H2O2 equivalent/l, and arbitrary unit respectively. CG, cancer group; HpeG, hyperthyroidism group; HpoG, hypothyroidism group; HG, healthy group.

Table 2

Estimation of the repeatability and reproducibility of each assay

Intra-assayDay-to-day
MeannCVi (%)MeannCVd (%)
TAS (mmol Trolox equiv./l)0.92201.750.93203.13
1.26201.221.30203.46
1.90201.792.03202.87
TOS (μmol H2O2 equiv./l)13.06202.0113.22202.76
16.88201.8918.72202.53
26.43201.9126.19202.56
FT3 (pg/ml)1.01204.61.96206.3
2.01202.83.64204.2
3.80201.79.79202.9
13.80201.413.05203.9
FT4 (ng/dl)0.39202.40.48234.8
0.90201.60.90313.7
1.47201.11.53322.9
3.00202.43.02313.3
6.36202.06.36303.2
TSH (mIU/l)0.17201.90.17215.1
10.4200.710.5211.6
36.1200.936.8212.2

One-way ANOVA showed (Table 1) significant differences among the study groups in the levels of oxidative stress markers TAS (F=16.958, P=0.000), TOS (F=55.796, P=0.000), and OSI (F=56.419, P=0.000), as well as thyroid profile TSH (F=813.033, P=0.000), FT3 (F=86.239, P=0.000), FT4 (F=191.500, P=0.000), and FT3/FT4 (F=37.327, P=0.000). These results indicated increased oxidative stress in both thyroid disease patients and thyroid cancer patients. However, no statistically significant differences were found in serum TAS, TOS, and OSI values among carcinoma histological subtypes (Table 3).

Table 3

Serum levels of oxidative stress markers in carcinoma histological subtypes

ItemsCancer (n)Means.d.Min.Max.Statistic values
TASPTC (53)1.380.201.041.70F=0.430, P=0.732
FTC (18)1.350.221.001.63
ATC (6)1.330.151.051.46
MTC (5)1.290.251.041.56
TTC (82)1.370.201.001.70
TOSPTC (53)21.812.8617.1826.55F=1.627, P=0.190
FTC (18)20.802.6016.2323.96
ATC (6)20.112.4916.7123.06
MTC (5)19.783.0116.5922.81
TTC (82)21.342.8216.2326.55
OSIPTC (53)1.610.311.142.33F=0.190, P=0.903
FTC (18)1.590.371.012.33
ATC (6)1.520.171.291.78
MTC (5)1.560.201.311.86
TTC (82)1.600.311.012.33

PTC, papillary thyroid cancer; FTC, follicular thyroid cancer patients; ATC, anaplastic differentiated thyroid cancer; MTC, medullary thyroid cancer; TTC, total thyroid cancer.

Multiple linear regression analysis were performed with the TAS, TOS, or OSI as dependent variables and using TSH, FT3, FT4, and FT3/FT4 as independent variables. The results showed no significant correlations between various oxidative stress markers and thyroid profiles for either the thyroid cancer patients or the controls (Table 4).

Table 4

Association between various oxidative stress markers and thyroid profiles by multiple linear regression analysis

nTSH (r, P)FT3 (r, P)FT4 (r, P)FT3/FT4 (r, P)RF, P
TAS50−0.046, 0.3760.034, 0.4080.104, 0.237−0.090, 0.2670.1420.230, 0.920
560.073, 0.2970.011, 0.469−0.011, 0.4670.082, 0.2740.1960.511, 0.827
82−0.031, 0.3910.106, 0.172−0.057, 0.3070.081, 0.2340.1270.315, 0.867
TOS50−0.004, 0.488−0.043, 0.3850.096, 0.254−0.128, 0.1880.1490.255, 0.905
560.114, 0.201−0.093, 0.2480.069, 0.307−0.015, 0.4560.3021.282, 0.289
82−0.036, 0.3760.132, 0.118−0.170, 0.0630.49, 0.0900.2751.578, 0.189
OSI500.069, 0.316−0.054, 0.356−0.034, 0.4080.005, 0.4860.1040.122, 0.974
560.032, 0.407−0.078, 0.2830.071, 0.301−0.084, 0.2700.2470.829, 0.513
82−0.002, 0.493−0.012, 0.458−0.049, 0.3300.008, 0.4710.1000.194, 0.941

r, Pearson's correlation coefficient; R, multiple correlation coefficient.

ROC curves for the abilities of TAS, TOS, and OSI to distinguish between benign and malignant diagnoses are plotted in Fig. 1A–D respectively. Based on the results from the ROC curve analysis, the optimal cutoff points were estimated using the maximum values of the Youden index (YI). AUC, cutoff values, sensitivity, specificity, and YI are reported in Table 5. The results showed that the OSI was the best indicator for distinguishing cancer patients from disease controls and/or healthy controls, followed by TOS and TAS. Compared with disease controls, AUC (95% CI) was 0.964 (0.933–0.994) for OSI, 0.929 (0.883–0.974) for TOS, and 0.702 (0.599–0.805) for TAS. Compared with healthy controls, AUC (95% CI) was 1.000 (1.000–1.000) for OSI, 0.996 (0.000–1.000) for TOS, and 0.878 (0.803–0.954) for TAS. Between disease controls and healthy controls, the AUC (95% CI) was 0.979 (0.960–0.998) for OSI, 0.957 (0.928–0.986) for TOS, and 0.776 (0.697–0.855) for TAS. These data show that lower TAS and higher TOS levels (i.e. increased oxidative stress) may be related to thyroid cancer.

Figure 1
Figure 1

ROC curve analyses of the oxidative stress biomarkers. (A) (HpeG+HpoG) vs CG; (B) HG vs CG; (C) (HG + HpeG+HpoG) vs CG; (D) HG vs (HpeG+HpoG). The TAS curve is inverted because the results are decreased.

Citation: Endocrine-Related Cancer 18, 6; 10.1530/ERC-11-0230

Table 5

Performance of oxidative stress markers in predicting thyroid cancer

AUC (95% CI)CutoffSeSpYI
(HpeG+HpoG) vs CG
 TAS0.706 (0.620–0.792)1.4185.748.80.345
 TOS0.963 (0.938–0.989)17.1792.785.70.784
 OSI0.975 (0.954–0.997)1.2190.294.60.848
HG vs CG
 TAS0.862 (0.801–0.923)1.5674.084.10.581
 TOS0.998 (0.000–1.000)16.5497.698.00.956
 OSI0.999 (0.000–1.000)1.1198.8100.00.988
(HG+HpeG+HpoG) vs CG
 TAS0.780 (0.715–0.845)1.4192.548.80.413
 TOS0.980 (0.965–0.994)16.6996.384.90.812
 OSI0.987 (0.974–0.999)1.1991.596.20.877
HG vs (HpeG+HpoG)
 TAS0.750 (0.659–0.841)1.5674.084.10.581
 TOS0.734 (0.639–0.829)16.8357.184.00.411
 OSI0.871 (0.803–0.939)0.9971.492.00.634

TAS, TOS, and OSI units are expressed as mmol Trolox equiv./l, μmol H2O2 equiv./l, and arbitrary unit respectively. Se, sensitivity; Sp, specificity; YI, Youden index; CG, cancer group; HpeG, hyperthyroidism group; HpoG, hypothyroidism group; HG, healthy group.

Risk estimate statistics were calculated using the cutoff values obtained by ROC curve analysis. These results showed that the TOS and/or TOS were sufficient risk factors to discriminate thyroid cancer from benign thyroid diseases and/or healthy controls (Table 6).

Table 6

Risk estimate for prediction of thyroid cancer using oxidative stress markers

TASTOSOSI
χ2ORχ2ORχ2OR
Paired groupsP95% CIP95% CIP95% CI
(HpeG+HpoG) vs. CG19.9904.9301659.14387.063135.785186.389
0.0002.362–10.2890.00066.433–104.0990.00055.025–631.367
(HpeG+HpoG+HG) vs. CG51.69410.3862235.779169.101182.089268.400
0.0005.070–21.2780.000123.786–231.0060.00087.145–826.651
HG vs. CG66.02814.5962262.7772225.061170.860a
0.0007.262–29.3380.000131.537–4020.0940.000
HG vs. (HpeG+HpoG)18.4854.166173.09714.72942.20727.547
0.0002.138–8.1180.0008.899–24.3780.0008.497–89.309

95% CI, 95% confidence interval; CG, cancer group; HpeG, hyperthyroidism group; HpoG, hypothyroidism group; HG, healthy group.

Risk estimate statistics cannot be computed because the specificity is 100%.

Discussion

Aerobic organisms have developed complex antioxidant systems that can counteract ROS and free radicals. This system can prevent and/or reduce ROS- and free radical-induced oxidative damage to tissues and cells (Wells et al. 2009) and is composed of different antioxidant substances, including the enzymes, nonenzymatic antioxidants, and an array of small molecules (Santi et al. 2010). These known and many unknown antioxidant substances compose the body's complex antioxidant system. To assess the antioxidant status in vivo, it is essential to measure the levels of overall antioxidants, including those known and unknown. In addition, the dynamic distribution of different antioxidants in various biological samples and their potential interactions make it difficult to measure each antioxidant separately, and such measurements are also unlikely to represent the overall antioxidant substances in the body. Similarly, to assess the oxidant status in vivo, it is essential to measure the levels of overall oxidants.

However, in previous studies concerning the role of antioxidants in thyroid cancer, most reports measured only one or several antioxidant substances separately (Senthil & Manoharan 2004, Akinci et al. 2008, Lin et al. 2009, Laatikainen et al. 2010, Young et al. 2010) such that the results did not reflect the total antioxidant levels in patients. In the current study, we recommended measuring the total oxidant/antioxidant status of an organism. Owing to the various interactions among antioxidants and oxidants, we believe that by measuring the total effects of the oxidants/antioxidants present in an organism, we can obtain more valuable results. Many previous studies have revealed increased oxidative stress in thyroid cancer (Abiaka et al. 2001, Senthil & Manoharan 2004, Alkhenizan and Hafez 2007, Akinci et al. 2008, Du et al. 2009, Lin et al. 2009, Laatikainen et al. 2010, Lassoued et al. 2010, Erdamar et al. 2010, Liu et al. 2010, Young et al. 2010). Thus, in our study, in addition to the TAS, we measured serum TOS levels and calculated OSI values. One-way ANOVA showed significant differences among study groups in the levels of TAS and TOS. However, no statistically significant differences were found in serum TAS, TOS, and OSI values among carcinoma histological subtypes. The OSI reflects the redox balance between oxidation and antioxidation in a subject in vivo. Our results showed that OSI values were decreased in thyroid disease patients, indicating that in these participants, the redox balance capability was poor and therefore oxidative stress was inevitable. Thus, OSI levels were lower in patients with thyroid cancer than in those with benign thyroid disease, which were in turn lower than those in the healthy subjects.

Thyroid hormones are associated with the oxidative and antioxidative status of an organism and thus play an important role in ROS and free radical production (Coria et al. 2009, Erdamar et al. 2010). In the thyroid, ROS and free radicals participate in physiological and pathological processes so that redox imbalance (Senthil & Manoharan 2004, Akinci et al. 2008, Laatikainen et al. 2010, Young et al. 2010). Antioxidative defense pathways of the organism play a crucial role in reducing the increased levels of free radicals generated by thyroid gland dysfunction (Carmeli et al. 2008). In hyperthyroidism, there is augmented production of ROS because the basal metabolic rate is increased, which in the presence of inadequate antioxidative defense results in the occurrence of oxidative stress in these patients (Saad-Hussein et al. 2011). In hypothyroidism, the basal metabolic rate is decreased and therefore a decrease in free radical production is expected because of the metabolic suppression caused by the lowered thyroid hormone levels (Erdamar et al. 2008). In thyroid cancer, cancerous thyroid tissues have accelerated lipid peroxidation and diminished antioxidant scavenging enzymes, leaving these tissues more vulnerable to the toxic effects of some free radical species (Erdamar et al. 2010). Our study, however, demonstrated no significant correlations between various oxidative stress markers and thyroid profiles using multiple linear regression analysis (Table 3). These results suggested that no direct association or not a linear relationship in serum between variations of the levels of thyroid hormones and redox imbalance were caused by thyroid gland dysfunction, although the thyroid hormone can regulate oxidative metabolism and affect enzymatic antioxidants such as SOD catalase, GPx, and glutathione reductase levels and non-enzymatic antioxidants such as vitamin E and C, glutathione, and uric acid levels in vivo. Possible explanations for this phenomenon include 1) a rigorous and complex antioxidant defense system in the body, 2) the impact on radical chain reactions (accelerated or delayed) by the altered levels of oxidants/antioxidants, 3) the partial effect on oxidant/antioxidants that are subject to change, or 4) other unknown causes to be investigated.

A previous study confirmed that markers of oxidative stress can be divided into three categories: 1) the formation of modified molecules by free radical reactions, 2) the consumption or induction of antioxidant molecules or enzymes, and 3) the activation or inhibition of transcription factors (Erdamar et al. 2010). The category of oxidative stress markers most likely to change when thyroid function is abnormal requires further study. However, should we need to explore what kind of antioxidants level is decreased? It is worth considering. To simply understand the oxidative stress status of patients, it is sufficient to measure the TAS and TOS and calculate the OSI. However, concerning the study of oxidative stress for thyroid cancer patients and/or benign thyroid disease patients, most researchers only determined the TAS (Mayer et al. 2004, Bacic-Vrca et al. 2005, Andryskowski & Owczarek 2007, Ish-Shalom et al. 2008, Torun et al. 2009), some also determined the TOS (Santi et al. 2010, Aslan et al. 2011), and very few studies also calculated the OSI (Aslan et al. 2011) values. However, the TAS had the least accuracy (both AUC and YI are minimal) in our study for the differential diagnosis of thyroid cancer from disease controls and/or healthy controls (Table 4 and Fig. 1). ROC analysis revealed that a perfect test would have an AUC of 1.000, sensitivity of 100%, a specificity of 100%, and a YI of 1.000 at a cutoff value of 1.01 OSI to distinguish thyroid cancer patients from the healthy population while both AUC and YI are maximal in other statistic groups (Table 4). These results suggest that the OSI may be a good candidate marker for distinguishing thyroid cancer from disease controls and/or healthy controls. Therefore, to obtain a comprehensive understanding of the oxidative stress status of a subject, we should not only measure the TAS and TOS but also calculate the OSI. It can be speculated that TAS is not practiced widely by researchers and clinicians because it reflects the oxidative stress with one-sidedness, i.e. the specificity is lower. Risk estimate statistics have shown that both TOS and OSI were better indicators than TAS either for discriminating thyroid cancer from controls (including disease and healthy controls) or for discriminating thyroid disease from healthy controls (Table 5). This result demonstrated once again that if we want to fully understand the oxidative stress status of patients in vivo, not only TAS and TOS measurements but also the calculation of OSI are necessary.

Recently, one of the focuses on the molecular pathogenesis of thyroid cancer is BRAFV600E mutation. BRAFV600E is a constitutively active onco-kinase and is the most common genetic alteration in PTC and ATC, albeit at a lower frequency (Nucera et al. 2011). BRAFV600E through the mitogen-activated protein kinase signaling pathway may control a network of genes crucial in integrating and regulating the extracellular and intracellular signaling in thyroid cancer cells. The BRAFV600E mutation in some studies has been significantly associated with extra-thyroidal extension, metastases, and recurrence in patients with PTC and ATC (Nucera et al. 2010). Recent investigations have suggested that oxidative stress caused by inflammation may be in part responsible for mutations of the BRAF gene (Martinez-Cadenas et al. 2011). Other authors also found that oxidative stress plays a critical role in inactivating mutant BRAF by geldanamycin derivatives (Fukuyo et al. 2008). These findings are similar to those of this study. However, the exact mechanism of BRAFV600E mutation induced by oxidative stress needs to explore.

Collecting tissue samples can cause injury, and the pre-analytical specimen treatment is complicated. Furthermore, it is difficult to develop the appropriate reference range, and the results may only reflect the local tissue levels in patients. In contrast, collecting blood samples can overcome these limitations. Therefore, we chose to use blood samples for the current study not only to facilitate scientific research but also to pave the way for future clinical application.

In this study, our observations suggest that the serum TOS and OSI values were higher and TAS levels were lower in patients with thyroid cancer than in controls. There were no correlations between thyroid profiles and oxidative/antioxidative parameters. Therefore, increased levels of oxidants and decreased levels of antioxidants may provide evidence for thyroid patients who were exposed to potent oxidative stress. OSI values may be more useful oxidative stress biomarkers than TAS and TOS for monitoring the clinical status of thyroid cancer. Further investigations are required to clarify the role of oxidative stress in the thyroid carcinogenesis. These results may shed light on the development of novel methods to distinguish thyroid cancer patients from disease controls and/or healthy controls.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The work was supported by the People's Republic of China Ministry of Science and Technology (2006AA020905) and BioSino Bio-technology and Science, Inc.

References

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    • Search Google Scholar
    • Export Citation
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    • Export Citation
  • AlkhenizanAHafezK2007The role of vitamin E in the prevention of cancer: a meta-analysis of randomized controlled trials. Annals of Saudi Medicine27409414. doi:10.4103/0256-4947.51459.

    • Search Google Scholar
    • Export Citation
  • AndryskowskiGOwczarekT2007The evaluation of selected oxidative stress parameters in patients with hyperthyroidism. Polskie Archiwum Medycyny Wewnetrznej117285289.

    • Search Google Scholar
    • Export Citation
  • AslanMCosarNCelikHAksoyNDulgerACBegenikHSoyoralYUKucukogluMESelekS2011Evaluation of oxidative status in patients with hyperthyroidism. Endocrine40285289. doi:10.1007/s12020-011-9472-3.

    • Search Google Scholar
    • Export Citation
  • AycicekAErelO2007Total oxidant/antioxidant status in jaundiced newborns before and after phototherapy. Jornal de Pediatria83319322. doi:10.2223/JPED.1645.

    • Search Google Scholar
    • Export Citation
  • AycicekAErelOKocyigitA2005Decreased total antioxidant capacity and increased oxidative stress in passive smoker infants and their mothers. Pediatrics International47635639. doi:10.1007/s12020-011-9472-3.

    • Search Google Scholar
    • Export Citation
  • Bacic-VrcaVSkrebFCepelakIMayerLKusicZPetresB2005The effect of antioxidant supplementation on superoxide dismutase activity, Cu and Zn levels, and total antioxidant status in erythrocytes of patients with Graves' disease. Clinical Chemistry and Laboratory Medicine43383388. doi:10.1515/CCLM.2005.069.

    • Search Google Scholar
    • Export Citation
  • BoelaertK2009The association between serum TSH concentration and thyroid cancer. Endocrine-Related Cancer1610651072. doi:10.1677/ERC-09-0150.

    • Search Google Scholar
    • Export Citation
  • CarmeliEBacharABarchadSMoradMMerrickJ2008Antioxidant status in the serum of persons with intellectual disability and hypothyroidism: a pilot study. Research in Developmental Disabilities29431438. doi:10.1016/j.ridd.2007.08.001.

    • Search Google Scholar
    • Export Citation
  • CoriaMJPastránAIGimenezMS2009Serum oxidative stress parameters of women with hypothyroidism. Acta Bio-Medica80135139.

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    • Search Google Scholar
    • Export Citation
  • ErdamarHDemirciHYamanHErbilMKYakarTSancakBElbegSBiberoğluGYetkinI2008The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status. Clinical Chemistry and Laboratory Medicine4610041010. doi:10.1515/CCLM.2008.183.

    • Search Google Scholar
    • Export Citation
  • ErdamarHCimenBGülcemalHSaraymenRYererBDemirciH2010Increased lipid peroxidation and impaired enzymatic antioxidant defense mechanism in thyroid tissue with multinodular goiter and papillary carcinoma. Clinical Biochemistry43650654. doi:10.1016/j.clinbiochem.2010.02.005.

    • Search Google Scholar
    • Export Citation
  • ErelO2004A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical Biochemistry37277285. doi:10.1016/j.clinbiochem.2003.11.015.

    • Search Google Scholar
    • Export Citation
  • ErelO2005A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry3811031111. doi:10.1016/j.clinbiochem.2005.08.008.

    • Search Google Scholar
    • Export Citation
  • FukuyoYInoueMNakajimaTHigashikuboRHorikoshiNTHuntCUshevaAFreemanMLHorikoshiN2008Oxidative stress plays a critical role in inactivating mutant BRAF by geldanamycin derivatives. Cancer Research6863246330. doi:10.1158/0008-5472.CAN-07-6602.

    • Search Google Scholar
    • Export Citation
  • HarmaMHarmaMErelO2005Oxidative stress in women with preeclampsia. American Journal of Obstetrics and Gynecology192656657. doi:10.1016/j.ajog.2004.07.085.

    • Search Google Scholar
    • Export Citation
  • Ish-ShalomSDurleshterLSegalENaglerRM2008Sialochemical and oxidative analyses in radioactive I131-treated patients with thyroid carcinoma. European Journal of Endocrinology158677681. doi:10.1530/EJE-07-0634.

    • Search Google Scholar
    • Export Citation
  • LaatikainenLECastelloneMDHebrantAHosteCCantisaniMCLaurilaJPSalvatoreGSalernoPBasoloFNäsmanJ2010Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer. Endocrine-Related Cancer17785796. doi:10.1677/ERC-10-0021.

    • Search Google Scholar
    • Export Citation
  • LassouedSMseddiMMnifFAbidMGuermaziFMasmoudiHEl FekiAAttiaH2010A comparative study of the oxidative profile in Graves' disease, Hashimoto's thyroiditis, and papillary thyroid cancer. Biological Trace Element Research138107115. doi:10.1007/s12011-010-8625-1.

    • Search Google Scholar
    • Export Citation
  • LinJCKuoWRChiangFYHsiaoPJLeeKWWuCWJuoSH2009Glutathione peroxidase 3 gene polymorphisms and risk of differentiated thyroid cancer. Surgery145508513. doi:10.1016/j.surg.2008.12.008.

    • Search Google Scholar
    • Export Citation
  • LiuBKuangAHuangRZhaoZZengYWangJTianR2010Influence of vitamin C on salivary absorbed dose of 131I in thyroid cancer patients: a prospective, randomized, single-blind, controlled trial. Journal of Nuclear Medicine51618623. doi:10.2967/jnumed.109.071449.

    • Search Google Scholar
    • Export Citation
  • Martinez-CadenasCBoschNPeñasLFlores-CouceEOchoaEMunárrizJAracilJPTajahuerceMRoyoRLozoyaR2011Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report. BMC Cancer11343doi:10.1186/1471-2407-11-343.

    • Search Google Scholar
    • Export Citation
  • MayerLRomićZSkrebFBacić-VrcaVCepelakIZanić-GrubisićTKirinM2004Antioxidants in patients with hyperthyroidism. Clinical Chemistry and Laboratory Medicine42154158. doi:10.1515/CCLM.2004.028.

    • Search Google Scholar
    • Export Citation
  • NuceraCLawlerJHodinRParangiS2010The BRAFV600E mutation: what is it really orchestrating in thyroid cancer?Oncotarget1751756. doi:10.1634/theoncologist.2010-0317.

    • Search Google Scholar
    • Export Citation
  • NuceraCNehsMANagarkattiSSSadowPMMekelMFischerAHLinPSBollagGELawlerJHodinRA2011Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer. Oncologist16296309.

    • Search Google Scholar
    • Export Citation
  • Saad-HusseinAHamdyHAzizHMMahdy-AbdallahH2011Thyroid functions in paints production workers and the mechanism of oxidative-antioxidants status. Toxicology and Industrial Health27257263. doi:10.1177/0748233710386409.

    • Search Google Scholar
    • Export Citation
  • SantiADuarteMMMorescoRNMenezesCBagatiniMDSchetingerMRLoroVL2010Association between thyroid hormones, lipids and oxidative stress biomarkers in overt hypothyroidism. Clinical Chemistry and Laboratory Medicine4816351639. doi:10.1515/CCLM.2010.309.

    • Search Google Scholar
    • Export Citation
  • SenthilNManoharanS2004Lipid peroxidation and antioxidants status in patients with papillary thyroid carcinoma in India. Asia Pacific Journal of Clinical Nutrition13391395.

    • Search Google Scholar
    • Export Citation
  • TorunANKulaksizogluSKulaksizogluMPamukBOIsbilenETutuncuNB2009Serum total antioxidant status and lipid peroxidation marker malondialdehyde levels in overt and subclinical hypothyroidism. Clinical Endocrinology70469474. doi:10.1111/j.1365-2265.2008.03348.x.

    • Search Google Scholar
    • Export Citation
  • UttaraBSinghAVZamboniPMahajanRT2009Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Current Neuropharmacology76574. doi:10.2174/157015909787602823.

    • Search Google Scholar
    • Export Citation
  • ValkoMLeibfritzDMoncolJCroninMTMazurMTelserJ2007Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry & Cell Biology394484. doi:10.1016/j.biocel.2006.07.001.

    • Search Google Scholar
    • Export Citation
  • WellsPGMcCallumGPChenCSHendersonJTLeeCJPerstinJPrestonTJWileyMJWongAW2009Oxidative stress in developmental origins of disease: teratogenesis, neurodevelopmental deficits, and cancer. Toxicological Sciences108418. doi:10.1093/toxsci/kfn263.

    • Search Google Scholar
    • Export Citation
  • YoungOCrottyTO'ConnellRO'SullivanJCurranAJ2010Levels of oxidative damage and lipid peroxidation in thyroid neoplasia. Head & Neck32750756. doi:10.1002/hed.21247.

    • Search Google Scholar
    • Export Citation
*

(D Wang and J-F Feng contributed equally to this work)

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    ROC curve analyses of the oxidative stress biomarkers. (A) (HpeG+HpoG) vs CG; (B) HG vs CG; (C) (HG + HpeG+HpoG) vs CG; (D) HG vs (HpeG+HpoG). The TAS curve is inverted because the results are decreased.

  • AbiakaCAl-AwadiFAl-SayerHGulshanSBehbehaniAFarghallyMSimbeyeA2001Serum antioxidant and cholesterol levels in patients with different types of cancer. Journal of Clinical Laboratory Analysis15324330. doi:10.1002/jcla.1045.

    • Search Google Scholar
    • Export Citation
  • AkinciMKosovaFCetinBSepiciAAltanNAslanSCetinA2008Oxidant/antioxidant balance in patients with thyroid cancer. Acta Cirúrgica Brasileira23551554. doi:10.1590/S0102-86502008000600013.

    • Search Google Scholar
    • Export Citation
  • AlkhenizanAHafezK2007The role of vitamin E in the prevention of cancer: a meta-analysis of randomized controlled trials. Annals of Saudi Medicine27409414. doi:10.4103/0256-4947.51459.

    • Search Google Scholar
    • Export Citation
  • AndryskowskiGOwczarekT2007The evaluation of selected oxidative stress parameters in patients with hyperthyroidism. Polskie Archiwum Medycyny Wewnetrznej117285289.

    • Search Google Scholar
    • Export Citation
  • AslanMCosarNCelikHAksoyNDulgerACBegenikHSoyoralYUKucukogluMESelekS2011Evaluation of oxidative status in patients with hyperthyroidism. Endocrine40285289. doi:10.1007/s12020-011-9472-3.

    • Search Google Scholar
    • Export Citation
  • AycicekAErelO2007Total oxidant/antioxidant status in jaundiced newborns before and after phototherapy. Jornal de Pediatria83319322. doi:10.2223/JPED.1645.

    • Search Google Scholar
    • Export Citation
  • AycicekAErelOKocyigitA2005Decreased total antioxidant capacity and increased oxidative stress in passive smoker infants and their mothers. Pediatrics International47635639. doi:10.1007/s12020-011-9472-3.

    • Search Google Scholar
    • Export Citation
  • Bacic-VrcaVSkrebFCepelakIMayerLKusicZPetresB2005The effect of antioxidant supplementation on superoxide dismutase activity, Cu and Zn levels, and total antioxidant status in erythrocytes of patients with Graves' disease. Clinical Chemistry and Laboratory Medicine43383388. doi:10.1515/CCLM.2005.069.

    • Search Google Scholar
    • Export Citation
  • BoelaertK2009The association between serum TSH concentration and thyroid cancer. Endocrine-Related Cancer1610651072. doi:10.1677/ERC-09-0150.

    • Search Google Scholar
    • Export Citation
  • CarmeliEBacharABarchadSMoradMMerrickJ2008Antioxidant status in the serum of persons with intellectual disability and hypothyroidism: a pilot study. Research in Developmental Disabilities29431438. doi:10.1016/j.ridd.2007.08.001.

    • Search Google Scholar
    • Export Citation
  • CoriaMJPastránAIGimenezMS2009Serum oxidative stress parameters of women with hypothyroidism. Acta Bio-Medica80135139.

  • DuZXZhangHYMengXGuanYWangHQ2009Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells. BMC Cancer95667. doi:10.1186/1471-2407-9-56.

    • Search Google Scholar
    • Export Citation
  • ErdamarHDemirciHYamanHErbilMKYakarTSancakBElbegSBiberoğluGYetkinI2008The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status. Clinical Chemistry and Laboratory Medicine4610041010. doi:10.1515/CCLM.2008.183.

    • Search Google Scholar
    • Export Citation
  • ErdamarHCimenBGülcemalHSaraymenRYererBDemirciH2010Increased lipid peroxidation and impaired enzymatic antioxidant defense mechanism in thyroid tissue with multinodular goiter and papillary carcinoma. Clinical Biochemistry43650654. doi:10.1016/j.clinbiochem.2010.02.005.

    • Search Google Scholar
    • Export Citation
  • ErelO2004A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical Biochemistry37277285. doi:10.1016/j.clinbiochem.2003.11.015.

    • Search Google Scholar
    • Export Citation
  • ErelO2005A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry3811031111. doi:10.1016/j.clinbiochem.2005.08.008.

    • Search Google Scholar
    • Export Citation
  • FukuyoYInoueMNakajimaTHigashikuboRHorikoshiNTHuntCUshevaAFreemanMLHorikoshiN2008Oxidative stress plays a critical role in inactivating mutant BRAF by geldanamycin derivatives. Cancer Research6863246330. doi:10.1158/0008-5472.CAN-07-6602.

    • Search Google Scholar
    • Export Citation
  • HarmaMHarmaMErelO2005Oxidative stress in women with preeclampsia. American Journal of Obstetrics and Gynecology192656657. doi:10.1016/j.ajog.2004.07.085.

    • Search Google Scholar
    • Export Citation
  • Ish-ShalomSDurleshterLSegalENaglerRM2008Sialochemical and oxidative analyses in radioactive I131-treated patients with thyroid carcinoma. European Journal of Endocrinology158677681. doi:10.1530/EJE-07-0634.

    • Search Google Scholar
    • Export Citation
  • LaatikainenLECastelloneMDHebrantAHosteCCantisaniMCLaurilaJPSalvatoreGSalernoPBasoloFNäsmanJ2010Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer. Endocrine-Related Cancer17785796. doi:10.1677/ERC-10-0021.

    • Search Google Scholar
    • Export Citation
  • LassouedSMseddiMMnifFAbidMGuermaziFMasmoudiHEl FekiAAttiaH2010A comparative study of the oxidative profile in Graves' disease, Hashimoto's thyroiditis, and papillary thyroid cancer. Biological Trace Element Research138107115. doi:10.1007/s12011-010-8625-1.

    • Search Google Scholar
    • Export Citation
  • LinJCKuoWRChiangFYHsiaoPJLeeKWWuCWJuoSH2009Glutathione peroxidase 3 gene polymorphisms and risk of differentiated thyroid cancer. Surgery145508513. doi:10.1016/j.surg.2008.12.008.

    • Search Google Scholar
    • Export Citation
  • LiuBKuangAHuangRZhaoZZengYWangJTianR2010Influence of vitamin C on salivary absorbed dose of 131I in thyroid cancer patients: a prospective, randomized, single-blind, controlled trial. Journal of Nuclear Medicine51618623. doi:10.2967/jnumed.109.071449.

    • Search Google Scholar
    • Export Citation
  • Martinez-CadenasCBoschNPeñasLFlores-CouceEOchoaEMunárrizJAracilJPTajahuerceMRoyoRLozoyaR2011Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report. BMC Cancer11343doi:10.1186/1471-2407-11-343.

    • Search Google Scholar
    • Export Citation
  • MayerLRomićZSkrebFBacić-VrcaVCepelakIZanić-GrubisićTKirinM2004Antioxidants in patients with hyperthyroidism. Clinical Chemistry and Laboratory Medicine42154158. doi:10.1515/CCLM.2004.028.

    • Search Google Scholar
    • Export Citation
  • NuceraCLawlerJHodinRParangiS2010The BRAFV600E mutation: what is it really orchestrating in thyroid cancer?Oncotarget1751756. doi:10.1634/theoncologist.2010-0317.

    • Search Google Scholar
    • Export Citation
  • NuceraCNehsMANagarkattiSSSadowPMMekelMFischerAHLinPSBollagGELawlerJHodinRA2011Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer. Oncologist16296309.

    • Search Google Scholar
    • Export Citation
  • Saad-HusseinAHamdyHAzizHMMahdy-AbdallahH2011Thyroid functions in paints production workers and the mechanism of oxidative-antioxidants status. Toxicology and Industrial Health27257263. doi:10.1177/0748233710386409.

    • Search Google Scholar
    • Export Citation
  • SantiADuarteMMMorescoRNMenezesCBagatiniMDSchetingerMRLoroVL2010Association between thyroid hormones, lipids and oxidative stress biomarkers in overt hypothyroidism. Clinical Chemistry and Laboratory Medicine4816351639. doi:10.1515/CCLM.2010.309.

    • Search Google Scholar
    • Export Citation
  • SenthilNManoharanS2004Lipid peroxidation and antioxidants status in patients with papillary thyroid carcinoma in India. Asia Pacific Journal of Clinical Nutrition13391395.

    • Search Google Scholar
    • Export Citation
  • TorunANKulaksizogluSKulaksizogluMPamukBOIsbilenETutuncuNB2009Serum total antioxidant status and lipid peroxidation marker malondialdehyde levels in overt and subclinical hypothyroidism. Clinical Endocrinology70469474. doi:10.1111/j.1365-2265.2008.03348.x.

    • Search Google Scholar
    • Export Citation
  • UttaraBSinghAVZamboniPMahajanRT2009Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Current Neuropharmacology76574. doi:10.2174/157015909787602823.

    • Search Google Scholar
    • Export Citation
  • ValkoMLeibfritzDMoncolJCroninMTMazurMTelserJ2007Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry & Cell Biology394484. doi:10.1016/j.biocel.2006.07.001.

    • Search Google Scholar
    • Export Citation
  • WellsPGMcCallumGPChenCSHendersonJTLeeCJPerstinJPrestonTJWileyMJWongAW2009Oxidative stress in developmental origins of disease: teratogenesis, neurodevelopmental deficits, and cancer. Toxicological Sciences108418. doi:10.1093/toxsci/kfn263.

    • Search Google Scholar
    • Export Citation
  • YoungOCrottyTO'ConnellRO'SullivanJCurranAJ2010Levels of oxidative damage and lipid peroxidation in thyroid neoplasia. Head & Neck32750756. doi:10.1002/hed.21247.

    • Search Google Scholar
    • Export Citation