Anomalii fetale, complicații ale sarcinii și tulburări de dezvoltare în copilărie asociate cu expunerea la erbicidul Agent Orange (acid 2,4,5-triclorofenoxiacetic [2,4,5-T], acid 2,4-diclorofenoxiacetic [2,4-D] și 2,3,7,8-tetraclorodibenzo-p-dioxină

Introduction

From 1961 to 1971, during the Vietnam war, the Uni­ted States military forces used herbicides over southern Vietnam for crop destruction over an estimated area of 3.6 million acres, with Agent Orange being the most used⁽¹⁾. Agent Orange, named after the colored stripe on the steel containers, represents an equal mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)⁽²⁾. Initial concerns over offspring harm date back to a 1969 report which mentioned that TCDD increased stillbirths and birth defects in pregnant rats, which were later seen in the Vietnamese population linked to the dioxin contaminant in Agent Orange^(1,3,4).

Properties and environmental persistence

2,4-D has been labeled since the 1940s as an effective herbicide against broadleaf weeds. Generally, it has low toxicity, it is eliminated through urine, and has a half-life of 18 to 23 hours⁽²⁾. 2,4,5-T contained the TCDD contaminant, which is a potent member of the dioxin family. TCDD is toxic even at low doses, determining multiple malformations such as cleft palate and hydronephrosis, which have been experimentally reproduced, and are mediated by aryl hydrocarbon receptors (AhR)⁽⁵⁾. An international consensus among health authorities indicates that dioxins can cause developmental and reproductive defects, and can also disrupt hormonal signaling⁽⁶⁾. When it comes to TCDD, its half-life ranges from hours to days or even years when it’s absorbed through soil. In adult humans, it is about 7 to 11 years, and 0.4 years in children; the difference is based on the amount of fatty tissue and its accumulation^(2,6). Even 40 years after the war ended, breast milk sample analysis from sprayed areas showed that mothers could pass these dioxins to their babies through breast milk, because dioxin contamination persisted in those regions^(7,8).

Toxicity mechanisms

TCDD is primarily mediated by the cytosolic AhR, a ligand-activated transcription factor that, once bound by dioxin, translocates to the nucleus and induces or suppresses a broad set of genes, altering cell-cycle progression, cellular adhesion and migration, receptor tyrosine kinase function, and nuclear hormone receptor and growth factor signaling⁽²⁾. Dioxin causes congenital disabilities by activating the AhR. Experiments have shown that genetically engineered animals that do not possess the AhR gene do not develop the typical dio­xin-induced malformation, since there are no receptors. In addition, dioxin can change the way genes are regulated through epigenetic mechanisms, particularly by disrupting DNA methylation, which is associated with congenital disabilities following maternal exposure during pregnancy^(2,5).

Pregnancy, malformations and effects

Recent studies have shown that TCDD suppresses the proliferation and migration of palatal mesenchymal cells and impairs their osteogenic differentiation. These effects appear through the activation of the AhR pathway and disruption of developmental regulations such as Maternally Expressed Gene 3 (MEG3) and the Transforming Growth Factor Beta /TGF-b/Smad signaling pathway, both responsible for cell growth regulation, differentiation, migration and maintaining normal embryonic development^(5,9,10).

Research studies analyzing the Seveso accident and its long-term effects on the second generation of exposed individuals found that the AhR gene modified the relationship between prenatal TCDD exposure and birth weight. Prenatal TCDD exposure was associated with altered thyroid hormone levels in the offspring and with increased childhood atopy^(11-14). Also, dioxin has been linked to reduced female fertility and reduced sperm quality, although the findings are inconsistent⁽²⁾.

American and Australian studies of male veterans provided little to no evidence of association on whether herbicide exposure is associated with decreased sperm counts or quality, subfertility, spontaneous abortion, stillbirth, neonatal or infant death, low birth weight, or preterm delivery⁽¹⁴⁾. A re-analysis of the Air Force Health Study also did not establish a robust association between paternal dioxin and adverse reproductive outcomes of conceptions⁽¹⁵⁾. On the other hand, a 2006 meta-analysis pooled thirteen Vietnamese and nine non-Vietnamese studies reported a positive association between parental exposure to Agent Orange and congenital disabilities, with a relative risk of 1.95⁽⁴⁾. Single-center Vietnamese data are consistent with a high anomaly burden: an overall congenital-anomaly prevalence of approximately 38 per 1000 live births, and a congenital heart disease birth prevalence substantially above pooled rates for comparable Asian low- and middle-income countries^(16,17).

Malformations related to Agent Orange discussed in clinical literature include spina bifida, cleft palate, limb deformities, structural heart disease and congenital hypothyroidism^(2,5). In the United States, an association between Agent Orange exposure and birth defects was not initially recognized, as the 1983 and 1988 studies conducted by the Centers for Disease Control and Prevention (CDC) did not demonstrate significant overall findings. However, the accumulation of subsequent evidence led to the recognition of spina bifida as a service-connected condition, along with other neural tube defects associated with Agent Orange exposure⁽¹⁾.

Recent evidence from studies conducted in the Vietnamese population exposed to Agent Orange-contaminated areas has raised concerns regarding functional neurodevelopment, as TCDD can be transferred from mothers to infants through breast milk. TCDD exposure may persist in maternal tissues and can be excreted during lactation, representing a potential route of postnatal exposure. In children born to mothers from heavily contaminated areas of Vietnam, higher perinatal TCDD levels were associated at 2 years of age with lower language scores in boys and with reduced gross motor scores in girls. Neonatal electroencephalography linked maternal dioxin exposure to altered brain electrical activity that predicted poorer neurodevelopment, and eye-tracking showed atypical gaze towards faces at 2 and 3 years old^(18-21). At 5 years old, cognitive and motor scores were lower in sprayed areas, and a correlation was found with attention-deficit and hyperactivity symptoms. At 8 years old, higher exposure was associated with poorer learning ability, with effects often stronger in boys^(22-25). Exposure to TCDD before birth or early life may have long-lasting effects: it can remain in the body for years and even influence later-life outcomes. It is also suggested that TCDD behaves differently from other related dioxins in how it affects the body^(26-29).

Several epidemiological studies conducted in Vietnamese children from Agent Orange-contaminated areas have investigated the long-term neurodevelopmental consequences of prenatal and early-life TCDD exposure. These studies used different neurodevelopmental assessment methods, including neonatal electroencephalography (EEG), eye-tracking, Bayley Scales of Infant Development, Movement Assessment Battery for Children (Movement ABC-2), and Kaufman Assessment Battery for Children (KABC-II). The main findings are summarized in Table 1.

Overall, these studies suggest that prenatal and early-life TCDD exposure may be associated with alterations in brain electrical activity, social gaze behavior, language development, motor function, cognitive performance and attention-related outcomes. Some effects appeared to be sex-specific, with several studies reporting stronger associations in boys.

Multigenerational and transgenerational concerns

Because TCDD is an endocrine disruptor with trans­ge­ne­rational effects seen in animal models, attention has shifted to possible effects in the grandchildren and great-grandchildren of those exposed. However, research on third- and fourth-generation victims remains inconclusive. In heavily exposed Vietnamese adults, it has been associated with altered reproductive and thyroid hormone levels and male sexual dysfunction^(30,31).

Limitations

The strongest human evidence comes from highly contaminated low-population cohorts, so the findings may not generalize to lower-exposed regions and victims, including Centers for Disease Control and Prevention-backed research into the Vietnam war veterans, where associations remain inconsistent. Also, evidence on multigenerational and transgenerational effects in humans remains preliminary and largely inconclusive.

Conclusions

This review was conducted to consolidate fragmented evidence regarding the developmental toxicity of Agent Orange, particularly the effects of its TCDD contaminant, on fetal growth, congenital abnormalities and childhood neurodevelopment. The rationale for this review is based on the continuing relevance of TCDD exposure more than five decades after Agent Orange use, as exposed populations may still experience long-term health consequences, and new epidemiological findings continue to emerge. By integrating mechanistic data, animal studies, epidemiological investigations and clinical evidence, this review provides a comprehensive overview of the current understanding of TCDD-related developmental effects.

However, several limitations should be acknowledged. The available evidence is heterogeneous due to differences in study populations, exposure assessment methods, geographic areas, follow-up periods and evaluated outcomes. In addition, establishing causal relationships remains challenging because of potential confounding factors, variations in co-exposures and the limited availability of long-term prospective data in some populations.

Despite these limitations, the accumulated evidence highlights the importance of continuous surveillance of exposed populations, particularly children born in contaminated areas. Understanding the developmental effects of TCDD may contribute to improved risk assessment, early identification of neurodevelopmental and congenital abnormalities, targeted medical follow-up, and preventive strategies to protect maternal and child health.

Autor corespondent:  Ina Pogonea E-mail: ina.pogonea@usmf.md

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.