Perfluoroalkyl substances (PFAS) and lead (Pb) as "cardiovascular disruptors" in 9-11-year-old children living in Syracuse, New York, United States.

OBJECTIVE: Per- and polyfluoro-alkyl substances (PFAS) and lead (Pb) are ubiquitous environmental toxicants with apparent impact on cardiovascular disease (CVD) risk. As one possible mechanism for this increased risk, we have previously demonstrated an association between Pb exposure and heightened cardiovascular reactivity to acute psychological stress, a CVD risk factor. The present study expands this approach and considers both PFAS and Pb exposures (and the possible interaction). METHODS: We assessed 14 serum PFAS and whole blood Pb concentrations in a sample of 9-11 year-old children (N = 291; 43.2% White, 56.8% Black; 53.5% female). We measured cardiovascular functioning at rest and during psychological stress as well as multiple indicators of subclinical CVD including resting blood pressure (BP), carotid-femoral pulse wave velocity (cfPWV), carotid intima-media thickness (cIMT), and left ventricular mass (LVM). Data analysis included general linear modeling as well as a non-parametric approach to study metal mixtures, specifically Bayesian Kernel Machine Regression (BKMR). RESULTS: Significant interactions between different PFAS and with Pb suggest the importance of considering toxicant mixtures when assessing potential disruption of the cardiovascular system. The pattern of findings suggests that greater "vascular reactivity" (elevated BP and vascular resistance during acute psychological stress) was associated with higher concentrations of perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), and Pb, but only when perfluorooctanoic acid (PFOA) was concurrently elevated. With respect to subclinical outcomes, increasing perfluorodecanoic acid (PFDA) was associated with greater cIMT (ß = 0.21, p = 0.010). CONCLUSION: To our knowledge this is the first study to consider how PFAS exposures might affect cardiovascular functioning and subclinical disease. Although a complex pattern of associations emerged, it does appear that PFAS and Pb can be classified as "cardiovascular disruptors" in children. Further research is needed to replicate these novel findings and determine whether these disruptions produce future cardiovascular disease.

Race, cortisol, and subclinical cardiovascular disease in 9- to 11-year-old children.

BACKGROUND: Non-Hispanic Black Americans have a greater risk for certain subtypes of cardiovascular disease (CVD; e.g., stroke and heart failure) relative to non-Hispanic White Americans. Moreover, Black relative to White adults consistently show elevated cortisol, a CVD risk. The impact of race, environmental stress, and cortisol on subclinical CVD has yet to be fully researched in children. METHOD: We assessed diurnal salivary cortisol slopes and hair cortisol in a sample of 9- to 11-year-old children (N = 271; 54% female) with roughly half self-identifying as either Black (57%) or White (43%). Two subclinical CVD indicators were assessed: carotid-femoral pulse wave velocity (cfPWV) and carotid intima-media thickness (cIMT). We assessed numerous environmental stress indicators. RESULTS: After adjusting for covariates, we found that Black children had significantly flatter diurnal cortisol slopes, higher hair cortisol, and thicker IMT than White children. Significant pathways were found: race → salivary cortisol slope → cfPWV (effect = -0.059, 95% CI [-0.116, -0.002]) and race → hair cortisol → cIMT (effect = -0.008, [-0.016, -0.002]). Black children also experienced significantly more environmental stress than White children; however, only income inequality served as a significant indirect pathway from race to salivary cortisol (effect = 0.029, [0.003, 0.060]). CONCLUSIONS: Relative to White children, Black children had significantly greater hair cortisol and flatter diurnal slopes which, in turn, were associated with greater subclinical CVD. As suggested by a significant indirect pathway, income inequality might partially explain the race-cortisol association. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Exposure to Arsenic and Subclinical Cardiovascular Disease in 9- to 11-Year-Old Children, Syracuse, New York.

Importance: Studies in adults have demonstrated associations between arsenic exposure and clinical and subclinical cardiovascular disease (CVD). No studies to date have considered potential associations in children. Objective: To examine the association between total urinary arsenic levels in children and subclinical indicators of CVD. Design, Setting, and Participants: This cross-sectional study considered 245 children, a subset from the Environmental Exposures and Child Health Outcomes (EECHO) cohort. Children from the Syracuse, New York, metropolitan area were recruited from August 1, 2013, until November 30, 2017, with enrollment throughout the year. Statistical analysis was performed from January 1, 2022, to February 28, 2023. Exposures: Total urinary arsenic was measured using inductively coupled plasma mass spectrometry. Creatinine concentration was used to adjust for urinary dilution. In addition, potential exposure routes (eg, diet) were measured. Main Outcomes and Measures: Three indicators of subclinical CVD were assessed: carotid-femoral pulse wave velocity, carotid intima media thickness, and echocardiographic measures of cardiac remodeling. Results: The study sample included 245 children aged 9 to 11 years (mean [SD] age, 10.52 [0.93] years; 133 [54.3%] female). The geometric mean of the creatinine-adjusted total arsenic level in the population was 7.76 µg/g creatinine. After adjustment for covariates, elevated total arsenic levels were associated with significantly greater carotid intima media thickness (ß = 0.21; 95% CI, 0.08-0.33; P = .001). In addition, echocardiography revealed that elevated total arsenic was significantly higher for children with concentric hypertrophy (indicated by greater left ventricular mass and greater relative wall thickness; geometric mean, 16.77 µg/g creatinine; 95% CI, 9.87-28.79 µg/g) relative to the reference group (geometric mean, 7.39 µg/g creatinine; 95% CI, 6.36-8.58 µg/g). With respect to exposure source, significant geographic clustering of total arsenic was found in 1 urban area of Syracuse, New York. Conclusions and Relevance: These findings suggest a significant association between arsenic exposure and subclinical CVD in children. Elevated total arsenic levels were found in an area of Syracuse with known elevations of toxic metals from industrial waste, suggesting historical pollution as a possible source. Given the novelty and potential importance of this association, further research is needed to confirm our findings. Any potential effect of urinary arsenic exposure in childhood on actual clinical CVD outcomes in adulthood remains to be determined.

Airborne levels of cadmium are correlated with urinary cadmium concentrations among young children living in the New York state city of Syracuse, USA.

Air pollution is a serious public health issue with early childhood exposure being of high concern because of the greater risk that children might experience negative health outcomes. Industrial sources in and near communities are one potential path of exposure that children might face with greater levels of air pollution correlating with higher levels of toxicants detected in children. We compare estimated ambient air concentrations of Cadmium (Cd) to a cohort (n = 281) of 9 to 11-year old children during their early childhood years (0-5 years of age) in a mid-size city in Upstate New York. Levels of Cd air pollution are compared to children's urine-Cd levels. Urine has been shown to be a superior biomarker to blood for Cd exposure particularly for longer-term exposures. We find that participants who reside in households that faced greater Cd air pollution during the child's early years have higher urine-Cd levels. This association is stable and stronger than previously presented associations for blood-Cd. Findings support expanded use of air modelling data for risk screening to reduce the potential health burden that industrial pollution can have.

Association of Sleep Quality With Greater Left Ventricular Mass in Children Aged 9 to 11 Years.

OBJECTIVE: Research has consistently found associations between sleep characteristics and cardiovascular disease risk in children, adolescents, and adults. Although primarily investigated in clinical samples (e.g., in those with sleep disorders), greater left ventricular mass is associated with poor sleep quality in nonclinical adult populations as well; however, this has not been evaluated in children or adolescents. Our study aim was to consider the relationship between objectively measured sleep characteristics and left ventricular mass in children. METHODS: We assessed sleep and cardiac structure in a biracial sample of 9- to 11-year-old children (n = 176; 41% White, 59% Black; 50% female). Sleep was assessed with actigraphy for five nights. Cardiac dimensions were assessed using echocardiography. RESULTS: After adjusting for covariates, we found that poor sleep quality was associated with significantly greater left ventricular mass (ß = 0.13, t(167) = 2.14, p = .034, Cohen d = 0.16, for activity during sleep; ß = 0.15, t(167) = 2.43, p = .016, Cohen d = 0.18, for sleep fragmentation). Other cardiac dimensions (namely, relative wall thickness and right ventricular dimension) were also significantly associated with sleep characteristics. Notably, associations did not differ as a function of sex or race. CONCLUSIONS: The present findings are novel and unique because no prior reports have systematically documented the association between poor sleep quality with potentially detrimental cardiac remodeling in a nonclinical sample of children. However, the novelty and importance of these findings require additional research for confirmation.

Linking metal (Pb, Hg, Cd) industrial air pollution risk to blood metal levels and cardiovascular functioning and structure among children in Syracuse, NY.

BACKGROUND: Exposure to air pollution has been linked to individual health effects in occupational environments and communities proximate to air pollution sources. Use of estimated chemical concentrations from the Risk Screening Environmental Indicators (RSEI) model, derived from the Toxics Release Inventory, can help approximate some contributions to individual lifetime exposure to risk from air pollution and holds potential for linkages with specific health outcome data. OBJECTIVES: Our objectives were: (1) use regression modeling to test for associations between observed blood metal concentrations in children and RSEI total air concentrations of the same metals released from proximate manufacturing facilities; (2) determine the relative contribution of RSEI air pollution to blood metal concentrations; and (3) examine associations between chronic metal exposure and cardiovascular functioning and structure in study participants. METHODS: Using data synthesis methods and regression modeling we linked individual blood-based levels of lead, mercury, and cadmium(Pb, Hg, Cd) and cardiovascular functioning and structure to proximate industrial releases of the same metals captured by the Environmental Protection Agency's (EPA) RSEI geographic microdata. RESULTS: We found that RSEI-derived ground-level ambient air concentrations of Hg and Cd were a significant predictor of blood metal levels, when controlling for covariates and other exposure variables. In addition to associations with blood metal findings, RSEI concentrations also predicted cardiovascular dysfunction and risk including changes in left-ventricular mass, blood pressure, and heart rate. DISCUSSION: Right-to-know data, such as EPA's RSEI, can be linked to objective health outcomes, rather than simply serving as a non-specific risk estimate. These data can serve as a proxy for hazard exposure and should be used more widely to understand the dynamics of environmental exposure. Furthermore, since these data are both a product of and contribute to regulatory decision making, they could serve as an important link between disease risk and translation-orientated national environmental health policy.

Dietary contributions to increased background lead, mercury, and cadmium in 9-11 Year old children: Accounting for racial differences.

BACKGROUND: Initial interest in the adverse consequences of exposure to lead (Pb), mercury (Hg), and cadmium (Cd) focused on relatively high exposures through environmental or occupational sources; however, recent evidence suggests even low-level background exposure to non-essential metals might be detrimental, particularly for children's health and development. One potentially important source of increased background levels of non-essential toxic metals is diet. OBJECTIVES: We considered whether differences in diet are associated with levels of non-essential metals in blood and whether racial differences in metals are mediated by dietary differences. METHODS: We assessed blood levels of Pb, Hg, and Cd in a sample of 9-11 year-old children (N = 295) comprised of 42% European Americans (EAs), 58% African American (AAs), and 47% female. Diet was assessed using 24-h dietary recalls during phone interviews administered to parents on two consecutive days (Friday and Saturday). The Healthy Eating Index-2105 (HEI-2015) was calculated to assess diet quality. RESULTS: The current study identified significant dietary sources of non-essential metal exposure - namely total fruit for Pb, total protein for Hg, and greens and beans for Cd. Moreover, AAs were found to have significantly higher blood levels of Pb and Hg than EAs and these racial differences were significantly mediated by these dietary differences. DISCUSSION: This study is one of very few to consider total diet in children and exposure to the non-essential metals Pb, Hg, and Cd, and the first to demonstrate that racial differences in increased background blood levels of non-essential toxic metals can be accounted for by racial differences in diet. Given regional differences in food consumption patterns and specific farm and store sources for the foods, the generalizability of the current findings has yet to be determined; however, commonly consumed foods appear to be a significant source of low-level non-essential metals.

Background lead and mercury exposures: Psychological and behavioral problems in children.

BACKGROUND: The potential harm from exposure to nonessential metals, particularly mercury (Hg) and lead (Pb), has been the focus of research for years. Initial interest focused on relatively high exposures; however, recent evidence suggests that even background exposures might have adverse consequences for child development. Identifying the extent of these consequences is now a priority. METHODS: We assessed blood Pb and Hg levels in a biracial sample of 9-11 year-old children (N = 203). Neurodevelopment and psychological functioning assessments included hostility, disruptive behaviors, emotion regulation, and autism spectrum disorder behaviors. Parasympathetic (vagal) responses to acute stress were indexed by heart rate variability (HRV) at rest and during stress. RESULTS: With increasing Pb levels, children exhibit higher levels of hostile distrust and oppositional defiant behaviors, were more dissatisfied and uncertain about their emotions, and had difficulties with communication. These significant associations were found within a range of blood Pb levels from 0.19 to 3.25µg/dL, well below the "reference value" for children of >5µg/dL. Vagal reactivity interacted with Hg such that increasing Hg was associated with increasing autism spectrum behaviors for those children with sustained vagal tone during acute stress. CONCLUSIONS: This study is the first to demonstrate an association between very low-level Pb exposure and fundamental psychological mechanisms that might explain prior associations with more complex outcomes such as delinquency. Analyses of vagal reactivity yielded entirely novel associations suggesting that Hg may increase autism spectrum behaviors in children with sustained vagal tone during acute stress. The novelty of these later findings requires additional research for confirmation and the cross-sectional nature of the data caution against assumptions of causality without further research.

Low-level mercury in children: associations with sleep duration and cytokines TNF-α and IL-6.

There is a sizeable literature suggesting that mercury (Hg) exposure affects cytokine levels in humans. In addition to their signaling role in the immune system, some cytokines are also integrally associated with sleep behavior. In this cross-sectional study of 9-11 year old children (N=100), we measured total blood Hg in whole blood, serum levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), and objectively measured sleep and activity using actigraphy. Increasing blood Hg was associated with significantly shorter sleep duration and lower levels of TNF-α. IL-6 was not associated with sleep or blood Hg. This study is the first to document an association between total blood Hg and sleep (albeit a small effect), and the first to consider the associations of total blood Hg with cytokines TNF-α and IL-6 in a pediatric sample. Further research using alternative designs (e.g., time-series) is necessary to determine if there is a causal pathway linking low-level Hg exposure to sleep restriction and reduced cytokines.

Immobilized metal affinity chromatography and human serum proteomics.

Metal-binding proteins have a pivotal role in normal and diseased states. We used metal affinity chromatography to enrich a fraction of human serum proteins on immobilized columns loaded with cadmium, nickel, zinc, copper, or lead in bis-Tris saline and these proteins were identified using LC-MS/MS. Tens of enriched proteins were identified and we here present the 20 most abundant for binding each metal. The binding of various proteins (complement C3, alpha-2-macroglobulin, serum albumin, apolipoprotein B-100, complement component 4B preproprotein, apolipoprotein A-I, serotransferrin, alpha-1-antitrypsin, ceruloplasmin, 47kDa protein, uncharacterized protein DKFZp686P15220, transthyretin, hemopexin, inter-alpha-trypsin inhibitor heavy chain H2, and histidine-rich glycoprotein) to different metals using immobilized metal affinity chromatography was compared to the literature. Although many metal-binding properties of these proteins have been confirmed, new metal-binding proteins have also been identified. The metal array use in the proteomic biomarker search technologies gives this data particular importance.

Fish consumption, low-level mercury, lipids, and inflammatory markers in children.

There is considerable evidence that consuming fish has numerous health benefits, including a reduced risk of cardiovascular disease. However, fish is also the primary source of human exposure to mercury (Hg). In a cross-sectional study of 9-11 year old children (N=100), we measured fish consumption, blood lipids, total blood Hg, diurnal salivary cortisol (4 samples collected throughout the day), and performed a proteomic analysis of serum proteins using spectral count shotgun proteomics. Children who consumed fish had a significantly more atheroprotective lipid profile but higher levels of blood Hg relative to children that did not consume fish. Although the levels of blood Hg were very low in these children (M=0.77 µg/L; all but 1 participant had levels below 3.27 µg/L), increasing blood Hg was significantly associated with blunted diurnal cortisol levels. Blood Hg was also significantly associated with acute-phase proteins suggesting systemic inflammation, and several of these proteins were found to significantly reduce the association between Hg and diminished cortisol when included in the model. This study of a pediatric population is the first to document an association between blood Hg, systemic inflammation, and endocrine disruption in humans. Without a better understanding of the long-term consequences of an atheroprotective lipid profile relative to blunted diurnal cortisol and systemic inflammation, a determination of the risk-benefit ratio for fish consumption by children is not possible.

Enzymatic detection of γ-hydroxybutyrate using aldo-keto reductase 7A2.

Gamma-hydroxybutyrate (GHB) is a prescribed medication as well as a drug of abuse. Its detection in various matrices for in-field forensic scientists remains a challenge. We have developed an assay that uses aldo-keto reductase 7A2 (AKR7A2) for the specific determination of GHB in various drinks. AKR7A2 was purified using Ni-affinity chromatography. The Michaelis-Menten constant for the GHB oxidation reaction was 10 mM, and the minimum detection limit was 4 mM. Ethanol was not a substrate for AKR7A2. In a coupled reaction with NADP(+), phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol, various beverages (orange juice, milk, soda, and numerous alcoholic drinks) containing GHB turned from blue to light yellow. In a second coupled reaction where diaphorase replaced PMS, the presence of GHB also caused the expected change of color in various beers.

Low-level Pb and cardiovascular responses to acute stress in children: the role of cardiac autonomic regulation.

OBJECTIVE: A number of studies suggest that Pb exposure increases cardiovascular disease risk in humans. As a potential mechanism for this effect, we recently reported a significant association between early childhood Pb levels and cardiovascular response to acute stress. The current study considers the association between current Pb levels and the autonomic nervous system activation pattern underlying the cardiovascular response to stress in a new cohort of children. METHODS: We assessed blood Pb levels as well as cardiovascular responses to acute stress in 9-11 year old children (N=140). Sympathetic activation (measured with pre-ejection period) and parasympathetic activation (measured with high frequency heart rate variability) were also assessed. RESULTS: In a sample with very low levels of blood Pb (M=1.0 µg/dL), we found that increasing blood Pb was associated with coinhibition of sympathetic and parasympathetic activation in response to acute stress. In addition, increasing Pb levels were associated with the hemodynamic stress response pattern typical of coinhibition--significantly greater vascular resistance and reduced stroke volume and cardiac output. CONCLUSIONS: Blood Pb levels were associated with significant autonomic and cardiovascular dysregulation in response to acute psychological stress in children. Moreover, these effects were significant at Pb levels considered to be very low and notably well below the 10 µg/dL, the Centers for Disease Control and Prevention definition of an elevated blood Pb level. The potential for autonomic dysregulation at levels of Pb typical for many US children would suggest potentially broad public health ramifications.

Effects of lead and mercury on the blood proteome of children.

Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry. Apolipoprotein E demonstrated an inverse significant association with lead concentrations (average being one microgram/deciliter) as deduced from LC-MS/MS and 2-D electrophoresis and confirmed by Western blot analysis. This coincides with prior findings that Apolipoprotein E genotype moderates neurobehavioral effects in individuals exposed to lead. Fifteen other proteins were identified by LC-MS/MS as proteins of interest exhibiting expressional differences in the presence of environmental lead and mercury.

Plasma prekallikrein levels are positively associated with circulating lipid levels and the metabolic syndrome in children.

Plasma prekallikrein (PK) has been shown to be associated with cardiovascular disease (CVD) and its risk factors, but these associations have not been investigated in children. The present study examined PK activity in relation to well-established cardiovascular risk factors in a cohort of children aged 9-11 years (N=97). We found a significant and positive association between PK and fasting levels of total cholesterol (p<0.01), non-high-density lipoprotein cholesterol (p<0.01), and triglycerides (p<0.001). In addition, there was a significant association between PK activity and the metabolic syndrome, a clustering of risk factors considered to have an impact on atherosclerosis and CVD mortality. Finally, we found that children with a family history of CVD had significantly elevated PK activity. These novel findings warrant further investigations into the relationship between circulating PK levels and CVD risk factors because PK may be involved in the progression of the disease state.

Neuronal localization of the mitochondrial protein NIPSNAP1 in rat nervous system.

The NIPSNAP (4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1) proteins belong to a highly conserved family of proteins of unknown function. We found that NIPSNAP1 binds to the branched-chain alpha-keto acid (BCKA) dehydrogenase enzyme complex, which is disrupted in maple syrup urine disease, a disease of branched-chain amino acid catabolism that results in neurological dysfunction. Phenylketonuric (PKU) and epileptic mice show altered expression of NIPSNAP1 in the brain. Therefore, the distribution and localization of NIPSNAP1 in rat brain was determined. Results show that NIPSNAP1 is expressed exclusively in neurons including pyramidal neurons in the cerebral cortex, Purkinje neurons in the cerebellum and motor neurons in the spinal cord. Dopaminergic neurons in midbrain and noradrenergic neurons in the brainstem, which are affected in PKU, also express NIPSNAP1. NIPSNAP1 is found to be localized in the mitochondrial matrix and can bind dihydrolipoyl-transacylase and -transacetylase components of the BCKA and pyruvate dehydrogenase complexes in vitro. Our data provide the first experimental evidence for a strictly neuronal expression of this mitochondrial protein in the rat nervous system.

Mitochondrial protein import and human health and disease.

The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease.

Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria.

Supporting a co-translational model of protein import into mitochondria, we have previously shown that ribosome-nascent chain complexes (RNCs) specifically bind to mitochondria. When producing RNCs using the rabbit reticulocyte lysate in vitro translation system, it was necessary to maximize ribosome loading with truncated nascent proteins because it had a direct impact on RNC binding. We describe here the optimal conditions for preparing RNCs. We show that translation temperature and reaction time are two critical factors, with 30 degrees Celsius and 15min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.

Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane.

The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.

Targeting proteins to mitochondria using TAT.

Disorders of mitochondrial function cause significant human disease and suffering. To date, correction of these mitochondria defects has depended on biochemical approaches and has not been achieved via gene therapies. Using previously described fusion proteins containing the transactivator of transcription (TAT) region from the HIV virus and green fluorescent protein (GFP), with and without a mitochondrial targeting sequence (MTS) from mitochondrial malate dehydrogenase (mMDH), we have investigated transduction across mitochondrial membranes. Both TAT-GFP and TAT-mMDH-GFP fusion proteins are protected from externally added protease when incubated with isolated mitochondria. Furthermore, both TAT fusion proteins rapidly enter cultured cells and transduce into mitochondria. However, the MTS allows processing of the fusion protein and is necessary for persistence in mitochondria over time. Neither degradation of import receptors nor disruption of the mitochondrial membrane potential or pH gradient inhibits protein transduction of either fusion protein. Furthermore, when injected into pregnant mice, TAT-mMDH-GFP is detectable throughout fetal and neonatal pups. These results indicate that TAT fusion proteins are able to traverse mitochondrial membranes through mechanisms that do not involve the regular import pathway, and that the addition of a MTS allows persistence of the fusion protein within mitochondria. TAT-MTS fusion proteins may represent a viable option as potential mitochondrial protein therapies.