The review, conducted by researchers at the Universidade Federal do Pará in Brazil, searched the Scopus, Web of Science, and PubMed databases for English-language articles published between 2014 and 2024, excluding reviews and meta-analyses. After removing duplicates from an initial pool of 414 articles and screening titles, abstracts, and full texts, 54 studies made the final cut. From these, the team extracted data on study objectives, main results, and recommendations for collection, storage, and biomarker detection. The overarching conclusion is clear: no single sample tells the whole story. Instead, laboratories should analyze multiple matrix types and multiple biomarkers, collect samples in duplicate, refrigerate or freeze them depending on storage duration, use preservatives, and ensure that staff are properly trained.

The central problem the review addresses is microbial fermentation. After death, bacteria and yeasts can convert glucose in the body into ethanol and carbon dioxide, a process that produces so-called post-mortem ethanol. This neoformation is not limited to visibly decomposing bodies; it can occur in specimens kept under inadequate conditions, such as high temperatures or the absence of preservatives like sodium fluoride. The result can be false-positive blood alcohol values that have nothing to do with what the deceased actually drank. Conversely, ethanol already present can be lost through oxidation into acetaldehyde, producing false negatives. Pathological conditions complicate matters further: individuals with diabetes, whose bodies carry excess glucose, can generate significant post-mortem blood ethanol even when they consumed no alcohol at all.

To disentangle drinking from fermentation, forensic toxicologists increasingly rely on non-oxidative metabolites of ethanol, chiefly ethyl glucuronide (EtG) and ethyl sulfate (EtS). Because these compounds are direct products of the body’s metabolism of alcohol, they cannot be manufactured by microbes fermenting glucose. Their presence in post-mortem samples is therefore directly tied to alcohol consumed before death. Studies cited in the review demonstrated a selective association between ante-mortem drinking and EtG and EtS detection: the biomarkers were absent in ethanol-negative samples from both deceased and living individuals, but appeared and then declined over time in ethanol-positive ones. Notably, EtG and EtS are not spontaneously formed even under post-mortem hyperglycemic conditions, so their detection supports metabolic activity before death, including in diabetic cases.

But the two biomarkers behave differently as bodies decompose. EtG is more sensitive yet less stable, and can be degraded during advanced decomposition, risking false negatives. EtS, by contrast, remains reliable even in severely putrefied blood, particularly at high temperatures, making it the more dependable marker for excluding pre-death drinking when results conflict. One strategy the review highlights is measuring EtG in vitreous humor, the clear fluid inside the eye, which is more sterile than blood and better protected from decomposition. Interpretation, the authors stress, must always account for the matrix, preservation conditions, and degree of decomposition.

Acetaldehyde, ethanol’s oxidative metabolite, is another piece of the puzzle, though a treacherous one. Its origin is difficult to pin down: it may arise from bacterial activity, yeast fermentation, ethanol oxidation, putrefaction, or poor blood preservation. The review notes that in well-preserved bodies and living individuals, acetaldehyde concentrations stayed within a stable range of 0.14 milligrams per milliliter or less, while highly putrefied bodies showed significantly higher values, indicating an association with putrefaction. Abnormally high acetaldehyde in the presence of ethanol and higher alcohols such as 1-propanol and 1-butanol may signal microbial neoformation. Conversely, acetaldehyde appearing without ethanol or 1-propanol may indicate that ethanol was oxidized and lost, particularly under extreme storage conditions. Higher alcohols themselves, produced when amino acids released by protein degradation ferment, appear at far lower concentrations than ethanol; a blood 1-propanol cutoff of 0.104 milligrams per deciliter has been proposed as a criterion for identifying post-mortem microbial ethanol production, though some researchers question its reliability.

When it comes to choosing samples, the review finds strong support for a multi-matrix approach. Peripheral blood, generally drawn from the femoral vein to avoid contamination and post-mortem redistribution, remains the primary specimen. Urine is closely associated with blood and, in healthy individuals, contains no glucose, reducing fermentation risk, though diabetic victims or those with urinary infections can confound this advantage. Vitreous humor emerges as a standout: anatomically isolated, minimally susceptible to decomposition and glycosuria, easy to collect from both eyes, and stable after death. Its inclusion as a routine matrix is strongly recommended, especially for putrefied bodies, outdoor deaths, and diabetic individuals. Cerebrospinal fluid, particularly from the spinal canal rather than the cerebral ventricles, offers another protected alternative. More exotic options are also on the table: dried blood spots for stable metabolites, costal cartilage ground in liquid nitrogen, brain tissue, hair for chronic abuse markers, and even necrophagous insect larvae, in which EtS proved a promising and stable indicator of alcohol ingestion.

Preservation practices emerge as the make-or-break factor in sample integrity. Refrigeration at 4 degrees Celsius is the minimum requirement, but samples stored longer than 48 hours should ideally be frozen, preferably at minus 80 degrees. Ethanol losses at room temperature can reach 15 to 16 percent, and even at minus 20 degrees, variations of 8 percent over 30 days have been recorded. Because oxidation is a primary cause of ethanol loss, samples should be collected in duplicate so that unopened tubes remain available for reanalysis; previously opened tubes showed greater losses in long-term studies. Tubes should be filled more than halfway to limit oxygen headspace, sealed tightly, made of sterile glass, and homogenized gently before storage. Fluoride preservatives, at concentrations of roughly 1 to 2 percent sodium or potassium fluoride, are essential: they inhibit both microbial growth and the enzyme enolase, curbing glycolysis and endogenous ethanol production. Anticoagulants such as EDTA or potassium oxalate may accompany them, while lithium heparin alone proved ineffective even at minus 80 degrees.

The stakes of all this extend well beyond the laboratory bench. Blood alcohol levels influence medicolegal interpretation, investigative conclusions, and judicial decisions, and the integrity of the evidence chain from collection to courtroom underpins due process itself. The review’s authors argue that protocols enabling clearer correlations between results and real conditions can prevent or detect alterations in ethanol levels, ensuring validity, traceability, and a fairer trial. They call for integrating ethanol metabolite analysis and multiple matrices into routine laboratory protocols, supported by standard operating procedures, investment in technology, and ongoing staff training. Emerging frontiers, from epigenetic markers of alcohol use disorder to ethyl esters revealing combined use of alcohol and synthetic cannabinoids, promise to widen the analytical toolkit further. For now, the message to forensic science is one of disciplined redundancy: sample widely, preserve rigorously, and never trust a single number to tell the story of how alcohol entered a body, or when.

Subject of Research: Post-mortem ethanol detection and the selection, collection, and storage of biological matrices in forensic toxicology

Article Title: Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage

Article References: da Paz, A. P. S., Nunez, L. B. B., Jr, & de Mello, V. J. (2026). Biological matrices for determining the origin of post-mortem ethanol: Recommendations for collection and storage. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03994-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03994-9

Keywords: forensic toxicology, post-mortem ethanol, ethyl glucuronide, ethyl sulfate, vitreous humor, microbial fermentation, sodium fluoride, blood alcohol, sample preservation, forensic autopsy, biomarkers, chain of custody

Fluoride occupies an unusual position in public health. Deliberately added to drinking water in controlled amounts to protect teeth, it also enters the environment through industrial processes ranging from phosphate fertilizer production to aluminum smelting, coal combustion, and the manufacture of glass, ceramics, and bricks. In regions with fluoride-rich minerals, well water can contain concentrations approaching 10 milligrams per liter, far above levels considered desirable. Food contributes as well: vegetables grown in contaminated fields, fish, and especially tea can carry appreciable fluoride loads, with dry tea leaves averaging around 100 milligrams per kilogram. Because the element is non-biodegradable, exposure is essentially continuous for many populations, making the search for practical protective strategies a matter of genuine urgency.

The biological problem with excess fluoride is that it behaves as a pro-oxidant. When fluoride intake overwhelms the body’s antioxidant defenses, free radicals accumulate faster than they can be neutralized, a state known as oxidative stress. This stress attacks the macromolecules of the cell, peroxidizing membrane lipids, damaging proteins, and breaking DNA strands. Previous studies in mice given sodium fluoride in drinking water showed depleted glutathione in the liver, along with DNA strand breaks, micronucleus formation, and the appearance of oxidized DNA adducts such as 8-hydroxy-2-deoxyguanosine. Such damage can push cells toward genotoxicity, somatic mutation, and apoptosis, the controlled self-destruction that follows irreparable injury. The liver, as the body’s principal detoxification organ, and the kidney, which receives roughly a fifth of cardiac output and concentrates circulating toxins, are particularly vulnerable targets.

To test whether Phyllanthus amarus could intervene, the team harvested leaves from Umuosele community in Amai, Nigeria, dried and powdered them, and extracted 500 grams of the material in methanol over 48 hours. The crude extract was then separated by liquid-liquid partitioning into fractions of differing polarity, using n-hexane, diethyl ether, n-butanol, methanol, and water. Yields varied considerably, with the methanol crude extract giving 37.62 percent, the methanol fraction 28.40 percent, and the aqueous fraction only 7.30 percent. The researchers selected the crude extract, the highest-yielding methanol fraction, and the lowest-yielding aqueous fraction for animal testing, dissolving each in 5 percent Tween 80 to a working concentration of 0.11 grams per milliliter.

Safety came first. In a preliminary lethal dose assessment involving sixty rats, no deaths occurred at oral doses from 100 to 1600 milligrams per kilogram of body weight across the fractions and crude extract. Rats receiving the higher doses showed reduced locomotion, but the 400 milligram per kilogram dose was well tolerated and even associated with normal hair growth, so the team adopted it for the main experiment. Thirty male Wistar rats, aged seven to nine weeks, were then divided into six groups of five. One group served as an untreated control, two groups received sodium fluoride alone or with the Tween 80 vehicle, and three groups received sodium fluoride alongside either the aqueous fraction, the methanol fraction, or the crude extract. Sodium fluoride was dosed orally at 25 milligrams per kilogram per day, and all treatments ran concurrently for 28 days before the animals were sacrificed and their tissues analyzed.

The results were striking. Rats exposed to sodium fluoride alone showed significant decreases in the activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, along with depleted levels of reduced glutathione, in both liver and kidney tissue. At the same time, malondialdehyde, the standard chemical marker of lipid peroxidation and membrane damage, rose significantly. Histological examination confirmed the biochemical picture: livers of fluoride-exposed rats showed necrosis, while kidneys displayed detachment of the glomerulus from Bowman’s capsule, clear signs of structural injury consistent with fluoride-induced oxidative stress.

Co-treatment with Phyllanthus amarus reversed much of this damage. In the groups receiving the aqueous fraction, the methanol fraction, or the crude extract, glutathione levels and the activities of all four antioxidant enzymes rose significantly compared with the fluoride-only animals, while malondialdehyde concentrations fell. The authors attribute this rescue effect to the plant’s rich polyphenol content, which can scavenge free radicals and thereby preserve the metal-dependent antioxidant enzymes that fluoride disrupts. Superoxide dismutase converts the superoxide anion into hydrogen peroxide, which catalase and glutathione peroxidase then eliminate, while glutathione-S-transferase conjugates electrophilic toxins for excretion. By keeping this enzymatic chain intact, the extracts appear to have restored the oxidative balance that fluoride had tipped.

Perhaps the most consequential finding concerned DNA. Using a diphenylamine-based fragmentation assay, the researchers measured how much DNA in liver and kidney tissue had been broken into pieces, a sensitive indicator of genotoxic injury. Fluoride-exposed rats showed markedly elevated DNA fragmentation in both organs, but the three treatment groups showed significant reductions. Notably, the crude extract outperformed the methanol fraction, a difference the authors suggest reflects the higher concentration of bioactive compounds present in the unfractionated preparation. The team proposes that the plant’s antioxidants reduce the electrophilic chemicals that would otherwise attack DNA, preventing the multiple lesions that fluoride toxicity can produce.

The study is not without limitations, which the authors acknowledge candidly. Financial constraints prevented characterization of the specific active constituents in the crude extract and fractions, so the precise molecules responsible for the protective effect remain unidentified. The work was also conducted in a small number of animals over a single 28-day window, and findings in rats do not automatically translate to humans. Nevertheless, the researchers argue that Phyllanthus amarus leaves merit further investigation as an affordable and accessible alternative for managing renal and hepatic disorders linked to fluoride and other oxidative stressors, particularly in regions where fluoride contamination of water and food is endemic.

For a plant that traditional healers have long prescribed for ailments ranging from hepatitis and malaria to diarrhea and skin disease, the new data provide a mechanistic footing for at least one of its claimed benefits. If subsequent studies can identify the active compounds and confirm protective effects at realistic exposure levels, a weed that grows freely across the tropics could become a low-cost ally against an environmental toxin that no one can entirely avoid. For now, the message from the Nigerian laboratory is clear: in rats at least, the chemistry of Phyllanthus amarus stands between fluoride and the fragile DNA of the liver and kidney.

Subject of Research: Protective effects of Phyllanthus amarus extracts against sodium fluoride-induced oxidative stress and DNA damage in rat liver and kidney

Article Title: Impact of Phyllanthus amarus on fragmented DNA and antioxidant activity of rats exposed to sodium fluoride

Article References: Ibobo, G. O., Okpoghono, J., & Onyesom, I. (2025). Impact of Phyllanthus amarus on fragmented DNA and antioxidant activity of rats exposed to sodium fluoride. Discover Toxicology, 2(1), Article 2. https://doi.org/10.1007/s44339-025-00017-x

Image Credits: AI Generated

DOI: 10.1007/s44339-025-00017-x

Keywords: Phyllanthus amarus, sodium fluoride, oxidative stress, DNA fragmentation, antioxidant enzymes, lipid peroxidation, malondialdehyde, glutathione, hepatotoxicity, nephrotoxicity, polyphenols, toxicology

The experimental design was deliberately comprehensive. Eighty-four adult male Wistar rats, each weighing between 100 and 120 grams, were randomly assigned to six groups. One group served as a control and received only deionized water, while the remaining five groups received sodium fluoride dissolved in deionized water at concentrations of 25, 50, 100, 150, and 300 parts per million, equivalent to 25 to 300 milligrams per liter. These doses deliberately exceed the World Health Organization guideline value of 1.5 milligrams per liter for fluoride in drinking water, but they fall within or above concentrations actually reported in groundwater from regions where fluorosis is endemic, making the findings directly relevant to millions of people worldwide who rely on fluoride-contaminated water sources. Seven rats from each group were evaluated at the 30-day mark and seven at 60 days, allowing the researchers to distinguish short-term from longer-term effects.

Behavioral testing using the open field assay revealed striking disturbances in both locomotion and anxiety-like behavior. In the 30-day exposure groups, rats receiving 25 milligrams per liter traveled significantly shorter distances and moved more slowly than controls, while animals at 50, 150, and 300 milligrams per liter showed significant reductions in maximum speed. Perhaps most tellingly, the treated rats exhibited disrupted movement patterns: increased angular velocity and meandering, reduced ability to maintain a straight path, and elevated anticlockwise rotation. Rats at the highest dose of 300 milligrams per liter showed markedly reduced freezing latency, suggesting an anxiety profile fundamentally different from controls. At 60 days, the pattern shifted, with some doses increasing distance traveled while others decreased mobility time, altered freezing behavior, and disrupted path efficiency, indicating that the behavioral consequences of fluoride exposure evolve over time rather than simply worsening in a linear fashion.

Beneath these behavioral changes, the biochemical assays painted a picture of severe oxidative stress in two critical brain regions, the hippocampus and the striatum. Malondialdehyde, a marker of lipid peroxidation and cellular membrane damage, rose in a dose-dependent fashion after 30 days of exposure. Hydrogen peroxide levels climbed while the brain’s antioxidant arsenal collapsed: reduced glutathione, superoxide dismutase, and glutathione peroxidase all declined significantly across treated groups. The brain is especially vulnerable to this kind of oxidative assault because it consumes enormous quantities of oxygen and is packed with polyunsaturated fatty acids that are prime targets for free radical attack. The researchers note that fluoride appears to suppress antioxidant defenses both directly, through competitive inhibition of protective enzymes, and indirectly, by generating a flood of reactive oxygen species that overwhelms them.

One of the most consistent findings across every dose and every time point was a significant reduction in acetylcholinesterase activity in both the hippocampus and the striatum. Acetylcholinesterase is the enzyme responsible for breaking down acetylcholine, a neurotransmitter central to learning, memory, attention, and motor control. Its inhibition means acetylcholine lingers longer at synapses, disrupting normal cholinergic transmission. This mechanism is pharmacologically significant because acetylcholinesterase inhibition is also the mode of action of certain nerve agents and pesticides, and reduced cholinergic transmission is implicated in cognitive decline. The authors suggest that the disrupted motor function and altered emotional states observed in the open field tests may be directly linked to this cholinergic impairment, providing a mechanistic bridge between molecular chemistry and observable behavior.

The immunohistochemical analysis added structural evidence to the biochemical story. In the striatum, the researchers documented elevated immunoreactivity for glial fibrillary acidic protein, or GFAP, a hallmark marker of astrocyte activation. Astrocytes normally support neurons by secreting neurotrophic factors, but when they proliferate excessively in response to injury, a process called astrogliosis, they can obstruct neurite outgrowth and impede tissue regeneration. After 60 days of exposure, the increase in astrocyte population was particularly pronounced. Gliosis is the brain’s intrinsic response to damage, and its presence here confirms that sodium fluoride exposure registers in the brain as genuine injury rather than a transient, reversible perturbation.

Equally concerning was the fate of the myelin sheath, the fatty insulation that wraps nerve fibers and enables rapid, efficient signal transmission. Rats exposed to sodium fluoride for both 30 and 60 days showed significantly reduced immunoreactivity for myelin basic protein in the corpus callosum, the massive fiber bundle connecting the brain’s two hemispheres. Demyelination is a serious form of neurological damage, characteristic of diseases such as multiple sclerosis, and its occurrence following chemical toxicity is well documented. The loss of myelin basic protein indicates breakdown of the intact myelin sheath, which would slow or scramble communication between brain regions and could plausibly underlie the motor coordination deficits and abnormal movement patterns recorded in the behavioral tests. Previous research has shown that fluoride can induce degenerative changes across the brain and spinal cord, including axonal degradation, mitochondrial impairment, and synaptic ultrastructural changes, and this study adds region-specific myelin loss to that inventory of harm.

Integrating these findings, the authors propose a coherent mechanistic chain: sodium fluoride exposure generates reactive oxygen species, depletes antioxidant defenses, and drives lipid peroxidation; this oxidative environment damages cholinergic neurons and triggers astrocytic proliferation; the resulting neuroinflammation and myelin degradation cumulatively degrade motor function, coordination, and anxiety regulation. The study’s strength lies in this multi-level convergence, linking behavioral observation with biochemical assay and structural histology in the same animals, something many earlier single-endpoint studies could not do. The connection to human health is not lost on the researchers, who note that epidemiological studies in regions with fluoride levels far exceeding WHO standards have reported significantly lower IQ scores in children, and that both juvenile and adult subjects exposed to elevated fluoride show diminished learning capacity and memory deficits.

The authors are careful to acknowledge the limitations of their work. All experiments were conducted in male rats, and since sex-specific differences in toxin susceptibility are widely reported, the findings may not fully generalize to females. Future studies incorporating female animals will be needed to determine whether these effects are sex dependent. The doses used, while environmentally relevant to fluorosis-endemic regions, sit well above typical fluoridated municipal water levels, so the results should not be read as an indictment of water fluoridation at recommended concentrations. Nevertheless, for the substantial populations in countries including Nigeria, India, Kenya, and China who drink groundwater with fluoride concentrations matching or exceeding those tested here, the implications are sobering. The study demonstrates that fluoride at these levels compromises brain health through oxidative stress, astrocytosis, and disrupted myelination, ultimately producing measurable neurobehavioral impairment, and it provides a detailed mechanistic framework that future protective interventions, whether antioxidant, cholinergic, or anti-inflammatory, can now be tested against.

Subject of Research: Neurotoxic effects of 30- and 60-day sodium fluoride exposure on behavior, brain chemistry, and myelin integrity in adult Wistar rats

Article Title: 30- and 60-days exposure of sodium fluoride induces neurobehavioral deficits and alters neurochemical parameters in adult Wistar rats

Article References: 30- and 60-days exposure of sodium fluoride induces neurobehavioral deficits and alters neurochemical parameters in adult Wistar rats. (n.d.). https://doi.org/10.1007/s44339-026-00051-3

Image Credits: AI Generated

DOI: 10.1007/s44339-026-00051-3

Keywords: sodium fluoride, neurotoxicity, oxidative stress, Wistar rats, acetylcholinesterase, myelin basic protein, GFAP, hippocampus, striatum, corpus callosum, neurobehavioral deficits, fluoridation