Ecotoxicology and Environmental Safety

Ecotoxicology and Environmental Safety 256 (2023) 114847

Available online 4 April 2023

0147-6513/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

Hypoxia inducible factor-1 α _(HIF-1 α ) as an early predictor of acute hydrogen cyanamide (Dormex) poisoning

Meriam N.N. Rezk

^a

, Gerges M. Beshreda

^b

, Dalia Abdelrahman Meshref

^c

,

Walaa Yehia Abdelzaher

^d

, Gaber El-Saber Batiha

^e

, Amin A. Hafiz

^f

, Duaa Althumairy

^g

, Nada H. Aljarba

^h

, Nermeen N. Welson

^i,*

aDepartment of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Minia University, Minia 61511, Egypt

bDepartment of Diagnostic Radiology, Faculty of Medicine, Minia University, Minia 61511, Egypt

cDepartment of Clinical Pathology, Faculty of Medicine, Minia University, Minia 61511, Egypt

dDepartment of Pharmacology, Faculty of Medicine, Minia University, Minia 61511, Egypt

eDepartment of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt

fDepartment of Clinical Nutrition, Faculty of Applied Medical Sciences, Umm Al, Qura University, Saudi Arabia

gDepartment of Biological Sciences, King Faisal University, Al-Ahsa 31982, Saudi Arabia

hDepartment of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia

iDepartment of Forensic Medicine and Clinical toxicology, Faculty of Medicine, Beni-Suef University, 62511 Beni Suef, Egypt

A R T I C L E I N F O Edited by Professor Bing Yan Keywords:

Hydrogen cyanamide Dormex

Hypoxia-inducible factor-1α _(HIF-1α₎ Follow up

A B S T R A C T

Hydrogen cyanamide (Dormex) is a plant growth regulator that is classified as a highly toxic poison. There are no definite investigations to help in its diagnosis and follow-up. This study aimed to investigate the role of hypoxia- inducible factor-1α (HIF-1α) in the diagnosis, prediction, and follow-up of Dormex-intoxicated patients. Sixty subjects were equally divided into two groups: group A, the control group, and group B, the Dormex group.

Clinical and laboratory evaluations, including arterial blood gases (ABG), prothrombin concentration (PC), the international normalized ratio (INR), a complete blood count (CBC), and HIF-1α, were done on admission. CBC and HIF-1α were repeated for group B 24 and 48 h after admission to track abnormalities. Group B also had brain computed tomography (CT). Patients with abnormal CT scans were referred for brain magnetic resonance im- aging (MRI). Significant differences in levels of HB, WBCs, and platelets were also detected in group B up to 48 h after admission, as white blood cells (WBCs) rose with time and hemoglobin (HB) and platelets diminished. The results described a highly significant difference in HIF-1α between the groups, and it depended on the clinical condition; therefore, it can be used in the prediction and follow-up of patients up to 24 h after admission.

1. Introduction

Cyanamide (CN2H2), in general, is an organic compound that is solid in consistency and has a white color. It is used mainly for agricultural and pharmaceutical purposes as well as the production of other com- pounds. It is composed of a nitrile and an amino group attached to each other. Many derivatives are there, like hydrogen cyanamide (CH2N2) and calcium cyanamide (CaCN2). Cyanamides are used as an alcohol deterrent in countries like Canada, Japan, and Europe (Güthner and Mertschenk, 2006). By causing aldehyde dehydrogenase deactivation,

calcium salts can enhance ethyl alcohol aversion (Glatt, 1959).

Dormex, the commercial name for hydrogen cyanamide, is widely used in agriculture to help uniform bud breaking in the spring and the blooming and early foliation of fruits. It helps farmers save the harvest that may be lost. It is usually used in the farms of blueberries, grapes, apples, peaches, kiwifruit, and, more recently, almonds and pistachios (Powell, 1999).

Hydrogen cyanamide has been enrolled in the red list of materials used in agriculture in Europe. The Environmental Protection Agency (EPA) has marked it as highly toxic (El-Alem et al., 2009; Occupational

* Corresponding author.

E-mail addresses: Meriamnabil23@gmail.com (M.N.N. Rezk), Scavenger120@gmail.com (G.M. Beshreda), meralmohamed2682013@gmail.com (D.A. Meshref), walaayehia22@yahoo.com (W.Y. Abdelzaher), gaberbatiha@gmail.com (G.E.-S. Batiha), aahafiz@uqu.edu.sa (A.A. Hafiz), Dalthumairy@kfu.edu.sa (D. Althumairy), Nhaljarba@pnu.edu.sa (N.H. Aljarba), nermeenwelson@med.bsu.edu.eg (N.N. Welson).

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

https://doi.org/10.1016/j.ecoenv.2023.114847

Received 18 December 2022; Received in revised form 15 March 2023; Accepted 28 March 2023

Safety and Health Administration (OSHA), 2001; Centers for Disease Control and Prevention (CDC), 2005).

However, it is still used in Egypt, especially in Minia, a city in Upper Egypt, to treat grape dormancy (Mohamed, 2008; Abbas et al., 2010).

Dormex has lamentable consequences for human health. Inhalational, dermal, and gastrointestinal absorptions are reported with different bioavailability. Exposure to hydrogen cyanamide vapor in the work- place is reported to be associated with dermal and respiratory irritation and headache. Some workers explained gastrointestinal manifestations like nausea, vomiting, or diarrhea (Schep et al., 2009).

Clinical presentations differ according to severity. Dermal manifes- tations include edema, erythema, dermatitis, and burns. Furthermore, dizziness, delirium, confusion, parathesia, and weakness are signs of neurological affection. Tachycardia, hypertension, dyspnea, and bron- chospasm are frequently reported. Coma, shock, and pulmonary edema

are also notable in severe cases of toxicity (Centers for Disease Control and Prevention (CDC), 2005; Gamaluddin et al., 2012). The explicit mechanism of action of Dormex is not well clarified. Moreover, there is no approved diagnostic test (Schep et al., 2009; Gamaluddin et al., 2012).

Hypoxia-inducible factor-1α (HIF-1α) is a primary factor that is responsible for regulating the body’s response to hypoxia. It enhances a cascade of changes in genes that mediate iron and glucose metabolism.

Cell multiplication, cell survival, and angiogenesis are also modulated by HIF-1α in low oxygen level conditions (Ke and Costa, 2006).

As there are no definite investigations to help in the diagnosis and follow-up, the aim of this study is to investigate HIF-1α in Dormex toxicity and estimate its role in the prediction of toxicity severity.

2. Subjects and methods 2.1. Study setting

This study was conducted from March 2022 to June 2022 in the Poison Control Center (PCC) of El Minia University Hospital. That period in Upper Egypt, especially in El-Minia city, is the known time for grape dormancy and the excessive use of Dormex to treat grape trees, which are available in abundance in El-Minia city.

2.2. Study design

The study was conducted on 68 subjects, from whom eight patients were excluded because they had underlying illnesses that could affect the results. The remaining subjects were divided into two groups: group A, the control group, which included 30 normal subjects, and group B, which included 30 Dormex toxicity patients. All Dormex-intoxicated patients who presented to the hospital in this grape dormancy season were included in the current cohort study.

2.3. Inclusion and exclusion criteria

Patients were diagnosed with Dormex poisoning according to the Platelets(£10³/

microliter) 285 (26.25) 221.5 (51.25) ˂˂0.001

PC % 79.50 ±9.02 79.58 ±9 0.97

INR 1.027 ±0.16 1.03 ±0.14 0.85

HIF-1α _{(IU) 0.41} ±0.13 2.65 ±0.34 ˂˂0.001

CT 0 ……. 24 (80%) ….

1 4 (13.33%)

2 1 (3.33%)

3 0 (0%)

4 1 (3.33%)

Numerical data were presented as the mean ±SD (standard deviation) and compared with independent t test or median (interquartile range; IQR) and compared with Mann-Whitney U test according to their normality. Categorical data were represented as the number and percentage. Significance is considered at P value <0.05. GCS: Glasgow coma scale, SBP: systolic blood pressure, DBP:

diastolic blood pressure, HR: heart rate, RR: respiratory rate, ABG: arterial blood gases, PCO2: carbon dioxide pressure, HCO3: bicarbonate, PO2: oxygen pres- sure, SO2: oxygen saturation, CBC: complete blood count, HB: hemoglobin, WBCs: white blood cells, PC: prothrombin concentration, INR: international normalized ratio, HIF-1α: hypoxia inducible factor 1 α, CT: computed tomography.

Table 2

Descriptive data of the Dormex group on admission.

Number (%)

Burn 1st degree 7 (23.33%)

2nd degree 23 (76.67%)

Brain injury Minor 8 (26.67%)

Moderate 17 (56.67%)

Severe 5 (16.67%)

Time of delay (hours) 11.83 ±4.95 ………

Data were presented as the mean ±SD (standard deviation) or the number and percentage.

Data were presented as the mean ±SD and compared with repeated measures ANOVA, or median (IQR) and compared with Friedman test. Significance is considered at P value <0.05. GCS: Glasgow coma scale, GCS1: Glasgow coma scale at time of admission, GCS 2: Glasgow coma scale at 24 h, GCS 3: Glasgow coma scale at 48 h, HB: hemoglobin, HB1: hemoglobin at admission, HB 2: he- moglobin at 24 h, HB 3: hemoglobin at 48 h, WBCs: white blood cells, WBCs1:

white blood cells at admission, WBCs2: white blood cells at 24 h, WBCs3: white blood cells at 48 h, Platelets1: platelets count at admission, Platelets2: platelets count at 24 h, Platelets3: platelets count at 48 h, HIF-1α: hypoxia inducible factor 1 α_{, HIF-1}α 1: hypoxia inducible factor 1 α at admission, HIF-1α 2: hypoxia inducible factor 1 α at 24 h, HIF-1α3: hypoxia inducible factor 1 α at 48 h.

history of exposure and the correlation of symptoms and signs of toxicity (Schep et al., 2009; Gamaluddin et al., 2012). Patients were asked to bring the container of the ingested substance if possible. Patients with a previous history of any neurological, respiratory, cardiac, renal, hep- atological, or hematological illness were excluded, in addition to pa- tients admitted 24 h after Dormex ingestion.

2.4. Ethical committee approval

The research was approved by the ethical committee board of the faculty of medicine at Minia University with approval number 305–2022. All procedures were explained to patients or their first-

degree relatives (depending on their level of consciousness). Enrolled patients were asked to sign informed consents if they agreed to participate.

2.5. Data collection 2.5.1. Clinical data

On admission, the clinical examination included the Glasgow coma scale (GCS), where patients’ eye, verbal, and motor responses were examined and given points according to the following criteria:

1-Eye Opening Response:

• Spontaneous eye opening with blinking at baseline: 4 points

• Responding to verbal commands and speech stimuli: 3 points

• Responding only to pain (not applied to the face): 2 points

• No response: 1 point.

2-Verbal Response:

• Oriented: 5 points

• Confused conversation, but able to answer questions: 4 points

• Inappropriate words: 3 points

• Incomprehensible speech: 2 points

• No response: 1 point 3-Motor response:

Fig. 1.Pattern of change in the measured parameters during the first 48 h of the disease course of the Dormex-intoxicated patients. A: HIF-1α, B: HB, C: WBCs, D: platelets.

Table 4

The correlations between HIF-1α measurements and GCS values.

Parameter R P value

GCS1& HIF-1α_{1 -0.801} ˂˂0.001

GCS2 & HIF-1α2 -0.737 ˂˂0.001

GCS3& HIF-1α3 -0.226 0.229

Correlations were made using the Spearman test. Significance is considered at P value <0.05. GCS: Glasgow coma scale, GCS1: Glasgow coma scale at time of admission, GCS 2: Glasgow coma scale at 24 h, GCS 3: Glasgow coma scale at 48 h, HIF-1α: hypoxia inducible factor 1 α, HIF-1α 1: hypoxia inducible factor 1 α at admission, HIF-1α 2: hypoxia inducible factor 1 α at 24 h, HIF-1α3: hypoxia inducible factor 1 α at 48 h.

•Obeying commands for movement: 6 points

•Purposeful movement to painful stimulus: 5 points

•Withdrawal in response to pain: 4 points

•Flexion in response to pain (decorticate posturing): 3 points

•Extension in response to pain (decerebrate posturing): 2 points

•No response: 1 point

The patients were classified as having a mild brain injury if their GCS was between 13 and 15 points, a moderate brain injury if their GCS was between 9 and 12 points, and a severe brain injury if their GCS was between 3 and 8 points (Jain and Iverson, 2020). GCS was assessed for all subjects in Group B 24 and 48 h after admission to track abnormal- ities. GCS 1 referred to GCS on admission; GCS 2 referred to GCS 24 h after admission; and GCS 3 referred to GCS 48 h after admission.

Furthermore, vital signs, including blood pressure (BP), heart rate (HR), temperature, and respiratory rate (RR), were recorded on admis- sion for all subjects. The presence or absence of an obvious skin burn was

noted.

2.5.2. Laboratory and radiologic investigations

Laboratory investigations included the measurement of arterial blood gases (ABG) using an AbG electrolyte analyzer (ST-200 alpha).

Prothrombin concentration (PC) and international normalized ratio (INR) were done using the LabiTec Gmbh semi-automated blood coag- ulation analyzer. Complete blood count (CBC) was performed using the Celltac G, Nihon Kohden Corporation, Automated Hematology Analyzer, Japan. The differential leukocytic count was confirmed by microscopic examination of a lieshman stained blood film (Houwen, 2002).

Hypoxia-inducible factor-1α (HIF − 1α) was measured by kits pur- chased from Eurofins Genomics, Europe. The total RNA was extracted from the blood samples and transcribed into complementary DNA using the GoTaq® 1-Step RT-qPCR System. Forward primer sequence: 5^′- TAT GAG CCA GAA GAA CTT TTA GGC − 3^′; reverse primer sequence: 5^′- CAC CTC TTT TGG CAA GCA TCC TG-3^′. The (2-ΔΔCt) formula was used to calculate the fold-change value of target genes in comparison to the control condition. For normalization, the reference gene GAPDH was used (VanGuilder et al., 2008).

Laboratory investigations, including ABG, PC, INR, CBC, and HIF-1, were done for every enrolled participant in groups A and B on admission.

CBC and HIF-1α were repeated for all subjects in the Dormex-intoxicated group 24 and 48 h after admission to track changes. Parameters were Fig. 2.Correlation of the measurements of HIF-1α to the values of GCS. a- Correlation between GCS and HIF-1α on admission. b- Correlation between GCS and HIF- 1α after 24 h. c- Correlation between GCS and HIF-1α after 48 h.

Table 5

ROC data of HIF-1α.

AUC Sensitivity Specificity (+ve)

predictive (-ve)

predictive Cutoff value

0.90 100.0% 68.0% 38.5% 100.0% 2.75

AUC: area under the curve.

divided into 1, 2, and 3, as parameter 1 referred to time on admission, parameter 2 referred to time 24 h after admission, and parameter 3 referred to time 48 h after admission.

Radiological investigations included brain computed tomography (CT), which was conducted by using a general electric light speed 16 detector CT, USA. Brain magnetic resonance imaging (MRI) was per- formed using a Phillips Ingenia 1.5 T MRI, Netherlands.

Radiological examinations included brain CT, which was performed for all patients in the Dormex-intoxicated group on admission, and brain MRI, which was performed only for patients with abnormal CT findings.

Findings of brain edema were recorded and scored as follows:

0 No cerebral edema

1 Focal edema limited to 1 lobe 2 Unilateral edema (>1 lobe) 3 Bilateral edema

4 Global edema (disappearance of sulcal relief)

5 Global edema (disappearance of sulcal relief +completely effaced basal cisterns) (Lietke et al., 2020).

2.5.3. Sample collection

For ABG, 1 cm of arterial blood was withdrawn with a pre- heparinized ABG syringe. Regarding the CBC, 1 cm of venous blood was withdrawn and kept in a lavender-colored top tube. For PC and INR, 1 cm of venous blood was put in a light-blue top tube (containing 3.2%

sodium citrate). One centimeter of blood was withdrawn from every patient and stored at − 80◦C for HIF-1α expression analysis.

2.6. Statistical analysis

SPSS version 26 was used to analyze the data. Numerical data were presented as the mean ±SD (standard deviation) and compared with independent t test or median and interquartile range and compared with Mann-Whitney U test according to their normality. Categorical data were represented as the number and percentage. Comparisons between the serial measures of the toxic group, on admission, 24 h and 48 h post admission, were made using Friedman test and Kruskal-Wallis test as a post-hoc. The Pearson test was used for correlation analysis of para- metric data and the Spearman test was used for the non-parametric data.

The receiver-operating characteristic curve (ROC) was used to assess the best cutoff point for the severity of toxicity with its sensitivity, speci- ficity, and area under the curve (AUC). A P value < 0.05, an alpha margin of error of 5%, and a 95% confidence interval were considered significant.

3. Results

This study included 60 subjects that fulfilled the inclusion criteria, and they were distributed into two groups; group A, the control group, contained 30 normal subjects, and group B contained 30 subjects with Dormex toxicity.

3.1. Comparison of groups A and B data on admission

The subjects enrolled were all males; their ages ranged between 26.87 ±5.51 for the control group and 25.77 ±5.03 for the diseased group (Table 1).

Regarding the clinical data, there were significant differences be- tween both groups in GCS, systolic blood pressure (SBP), and RR with a p value less than 0.001. GCS and SBP were noticed to be higher in the control group than the diseased group; however, RR was significantly increased in the diseased group.

Investigational data showed that the partial pressure of carbon di- oxide (PCO2), bicarbonate (HCO3), partial pressure of oxygen (PO2), Fig. 3.Receiver operating characteristic (ROC) curve of hypoxia inducible factor 1 α _(HIF-1α) in group B. The cutoff value of HIF-1α1 was 2.75 IU to diagnose Dormex toxicity with 100% sensitivity and 68% specificity.

Table 6

Correlations of HIF-1α to other measured parameters.

Parameters R p value

PCO2 0.26** 0.17

HCO3 -0.43* 0.02

HB 0.29* 0.13

WBCs 0.19* 0.31

Platelets 0.01** 0.94

Pearson correlation test* was performed for parametric data and Spearman test** for the non-parametric data. Significance is considered at P value <0.05.

PCO2: carbon dioxide pressure, HCO3: bicarbonate, HB: hemoglobin, WBCs:

white blood cells.

Table 7

CT brain edema scores in group B.

Parameter Score Number (%)

Brain CT edema score 0 24 (80%)

1 4 (13.33%)

2 1 (3.33%)

3 0 (0%)

4 1 (3.33%)

5 0 (0%)

Data were described as the number and percentage. CT: computed tomography.

Fig. 4. Grade 4 brain edema in Brain computed tomography (CT) of a Dormex-intoxicated patient.

oxygen saturation (SO2%), white blood cells (WBCs), platelets, and HIF- 1α showed differences between the two groups (p ˂0.001). PCO2, HCO3,

PO2, SO2%, and platelet measurements were elevated in the control group.

On the contrary, WBCs and HIF-1α were higher in the diseased group than the control group (Table 1).

3.2. Data for group B (Dormex group)

On admission, 23 patients in the Dormex-intoxicated group suffered from skin or mucous membrane burns. Eight, seventeen, and five pa- tients, respectively, presented with minor, moderate, and severe brain injuries. The time between exposure and admission was about 11.83 ± Fig. 5. Spearman correlation of GCS and CT score on admission, r= −0.67 &P-Value <0.001. GCS: Glasgow coma scale on admission, CT: computed tomography.

Fig. 6. An MRI film of a Dormex-intoxicated patient illustrated bilateral fronto-occipital periventricular and deep white matter abnormal signals with symmetrical appearance, high diffusion-weighted imaging (DWI) signals, and a corresponding low signal on the apparent diffusion coefficient (ADC) map, suggestive of mild restricted diffusion. They displayed faint, high-flair signals in these areas. The abnormal areas were hypo to isointense at T1W images indicating features of deep white matter disorder. Brain MRI reports revealed evidence of leukoencephalopathy.

(Table 4 and Fig. 2). The cutoff value of HIF-1α1 was 2.75 IU to diagnose Dormex toxicity with 100% sensitivity and 68% specificity (Table 5 and Fig. 3).

3.5. Correlations of HIF-1α to other measured parameters

There was a moderate negative correlation between HIF-1α level and HCO3 value. But no other significant correlations were detected (Table 6).

3.6. Radiological examination of group B (Dormex group) patients The brain CT results described 24 (80%) patients with normal brain CT. Brain edema with different degrees according to the scoring system was recorded in 6 (20%) cases (Table 7) (Fig. 4).

Brain CT showed a negative correlation to GCS on admission (r= − 0.586, P=0.001) (Fig. 5). Brain MRI was done in cases with grade 4 brain edema, and the report revealed evidence of bilateral fronto-occipital periventricular and deep white matter abnormal signals with symmetrical appearances suggestive of mild restricted diffusion.

Brain MRI reports revealed evidence of leukoencephalopathy (Fig. 6).

4. Discussion

Hydrogen cyanamide (CH2N2), the chemical responsible for the toxicity associated with Dormex ingestion, was first used in plant treatment in 1984. Dormex is a blue-colored solution that is applied to grape trees to cure their dormancy and aid in bud break (Bradbury, 2007). It is classified as having a high level of toxicity (Centers for Disease Control and Prevention (CDC), 2001). Our knowledge about its health burden is still not well clarified (Environmental Protection Agency (EPA), 2003).

In the current study, there were 60 patients enrolled, with 30 pa- tients in each group. The subjects enrolled were all males, and their ages ranged between 26.87±5.51 for the control group and 25.77±5.03 for the diseased group. It is easily explained as all toxicities were occupational in farms in Upper Egypt, where adult young males are preferred as workers.

The current results displayed a highly significant difference between the two groups in the GCS, SBP, and RR readings. GCS and SBP were higher in the control group than the diseased group, despite the fact that SPB was still within its normal range on admission in the diseased group (Verdecchia, 2001). RR was significantly higher in the diseased group.

In accordance with these findings, Gamaluddin and his colleagues examined 12 patients with Dormex toxicity who suffered from tachyp- nea, coma, and hypotension up to shock in 50% of cases (Gamaluddin et al., 2012). Also, Sharif et al. studied 35 cases with Dormex ingestion and stated that the declines in GCS and blood pressure were more obvious among patients with an unfavorable diagnosis (Sharif and

which can result in uncoupling oxidative phosphorylation and decreased adenosine nucleotide production. The downregulation of aldehyde de- hydrogenase causes a disulfiram-like syndrome characterized by hypo- tension, tachycardia, and confusion (Manoilov et al., 1996; De Haro, 2009; Cederbaum and Dicker, 1985).

Dormex-intoxicated patients in the present study had lower PCO2,

HCO3, PO2, and SO2% readings than the control group; however, PO2 and SO2% readings did not decline to critical values. This can refer to the presence of metabolic acidosis and respiratory system affection (Altalag et al., 2019). The same results were also described in previous human studies on Dormex poisoning (Gamaluddin et al., 2012; Sharif and Fayed, 2021). Sharif et al. described that the pH and HCO3 values were significantly dropped in cases with unfavorable outcomes; however, they reported only 6 cases among the total 35 patients with metabolic acidosis (Sharif and Fayed, 2021).

That effect could be illustrated by cellular inhibition of aerobic metabolism, cellular dependence on anaerobic metabolism, and decreased oxygen delivery to cells that elevate lactic acid production (Manoilov et al., 1996).

The current results revealed that the platelet count decreased and WBCs increased at the time of admission for the diseased group. Despite that, the platelet and WBC readings were still within their normal ranges (Tefferi and Hanson, 2005). The results of the current study are totally consistent with Gamaluddin et al., who stated the elevations in WBCs and declines in platelet count among his patients (Gamaluddin et al., 2012). Also, Sharif and his colleagues illustrated significant thrombo- cytopenia in association with an elevated leukocyte count among pa- tients with unfavorable outcomes (Sharif and Fayed, 2021).

Our follow-up results for the intoxicated group showed significant differences in levels of HB, WBCs, and platelets on admission, 24 h after admission, and 48 h after admission. WBC levels were noticed to be rising with time; however, patients’ HB and platelet readings showed a decline.

A well-established explanation for leukocytosis is Mitani et al.’s, who connected leukocytosis with the inflammatory body response due to high levels of eosinophils and leukocytes after Dormex exposure (Mitani et al., 2005). Gloxhuber et al. reported the presence of circulating cy- anamide serum antibodies in 72% of workers dealing with Dormex (Gloxhuber et al., 1989). Thrombocytopenia here is well clarified by previous studies, which linked the decline of thrombopoietin to liver and renal dysfunction (Gamaluddin et al., 2012; Sharif and Fayed, 2021).

Thrombopoietin is responsible for the stimulation of bone marrow for platelet synthesis (Cheung et al., 2005). Anemia that develops with follow-up can be explained by the prolonged coma and decreased oral feeding in addition to the corrosive action of Dormex, which develops difficult swallowing in most cases (Walsh et al., 2006; Colom et al., 1999).

The current study tried to reveal the role of HIF-1α in the mechanism of Dormex toxicity in humans. Our results disclosed a high level of HIF-

1α in the diseased patients. By following up with the hospitalized pa- tients, HIF-1α levels declined with treatment; however, their level did not fall to normal up to 48 h after admission.

Many previous studies were done to distinguish the role of HIF in other cases of hypoxia. It is considered the main factor regulating cellular hypoxic conditions. HIF is primarily made up of HIF-1β and HIF- 1α subunits, with HIF-1α being an oxygen sensitive subunit, as well as HIF-2α and HIF-3α paralogs (Ke and Costa, 2006). When hypoxia is initiated, HIF starts to activate the genes responsible for glucose trans- portation, the enzymes responsible for glycolysis, erytheropiotein, and other different genes responsible for stabilizing oxygen delivery and enhancing the adaptation of metabolism in low oxygen conditions (Semenza, 2000).

In the present results, the HIF-1α level was negatively correlated to GCS and HCO₃ readings. There were strong negative correlations be- tween HIF-1α1 and HIF-1α2 levels and GCS1 and GCS2 scores, respec- tively. On the contrary, a negative but not significant correlation between HIF-1α3 and GCS3 was noted. So it was noticed that HIF-1 was higher in deeply comatose patients. To help physicians select cases for ICU admission or tertiary hospital referral, the cutoff value for HIF-1α was calculated to be 2.75 IU on admission. It offers a non-subjective method of Dormex-intoxicated patients’ evaluation and can also be used to follow up on patients’ conditions up to 24 h after admission.

Per our knowledge, the current study is the first to distinguish the role of HIF-1α in the Dormex mechanism of toxicity and its role in the prediction of the need for referral or ICU admission in cases of Dormex exposure, in addition to their conditions’ follow-up. Hypoxia-inducible factor elevation in Dormex-intoxicated patients helps to clarify the mechanism of action of Dormex, which is still unclear, and emphasizes the theory of the existence of cellular hypoxia, which enhances its secretion. Physicians rely on history and clinical examination to di- agnose Dormex. HIF-1α measurement can help in Dormex coma differ- entiation, particularly in the absence of history.

Radiological examinations of our cases revealed the presence of brain edema in 20% of cases, varying in severity.

The brain CT correlation to the GCS value on admission showed a significant negative correlation between both parameters. Brain MRI in patients with grade 4 brain edema revealed mildly restricted diffusion and evidence of leukoencephalopathy. Our results matched those of El Mahdy and his colleagues, who reported a case of severe Dormex intoxication with evident CT brain edema (El Mahdy and Kharoub, 2020).

5. Conclusion

Hydrogen cyanamide poisoning is a devastating health burden that affects an extended sector of agriculture workers in Egypt, especially with the lack of knowledge and safety measurements. This study proves the hypothesis about cellular hypoxia associated with Dormex toxicity by measuring HIF − 1 α which helps in future treatment modulation, especially in the absence of an effective antidote. Early assessment of HIF − 1 α level can be of predictive value for the need for ICU admission and tertiary hospital referral, as most ingestion accidents occur in rural areas. HIF − 1 α at a level of 2.75 IU or more is an indicator of severe toxicity on admission, and its follow-up is beneficial for 24 h after admission. Testing of HIF-1α is a promising tool in Dormex coma diag- nosis, especially in the absence of a confirming diagnostic test. A com- plete blood count follow up is mandatory in all Dormex poisoning cases.

Recommendations

In doubtful cases, hypoxia-inducible factor − 1α can be used to differentiate the presence of Dormex toxicity. The hypoxia-inducible factor − 1 α level of 2.75 IU or more in Dormex poisoning is an indi- cator of ICU admission. The use of HIF-1α for patient follow-up in the first 24 h is beneficial, as it declines when patients start to improve. A

complete blood count follow-up is mandatory in Dormex toxicity as platelet measurements decline and WBCs rise even if the first readings are normal.

Agricultural workers should be enrolled in campaigns to increase their awareness of the disastrous health results and learn how to protect themselves. The search for more safe alternatives is crucial. More studies are needed to establish confirmative diagnostic tests and set a suitable treatment regimen, especially after clarifying the mechanism of action.

More studies are needed to reveal the role of brain MRI in the diagnosis of neurological insults associated with Dormex ingestion.

Ethical statement

The research was approved by the ethical committee board of the faculty of medicine, Minia University, with approval number 305-2022.

All procedures were explained to patients or their first-degree relatives (depending on their level of consciousness). Enrolled patients were asked to sign informed consents if they agreed to participate.

CRediT authorship contribution statement

Meriam N N Rezk: conceptualization, data collection, Writing – original draft, Gerges M Beshreda: conceptualization, radiological re- ports, Dalia Abdelrahman Meshref: laboratory investigations, Walaa Yehia Abdelzaher: Writing – original draft, Gaber El-Saber Batiha:

Writing – reviewing and editing, Amin A Hafiz: project administration, Duaa Althumairy: methodology, Nada H. Aljarba: resources, software, project administration, Nermeen N. Welson: methodology, data anal- ysis, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgment

The authors acknowledged the funding support of Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R62), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

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