Stem cell and benzene-induced malignancy and hematotoxicity

The biological effect of benzene on the hematopoietic system has been known for over a century. The rapid advancement in understanding the biology of hematopoietic stem cells (HSCs) and cancer stem cells (CSCs) in recent years has renewed interest in investigating the role of stem cells in benzene-induced malignancy and bone marrow depression. The interplay between benzene and stem cells is complex involving the stem cell, progenitor, and HSC niche compartments of the bone marrow. In this prospect, benzene metabolites formed through metabolism in the liver and bone marrow cause damage in hematopoietic cells via multiple mechanisms that, in addition to traditionally recognized chromosomal aberration and covalent binding, incorporate oxidative stress, alteration of gene expression, apoptosis, error-prone DNA repair, epigenetic regulation, and disruption of tumor surveillance. However, benzene-exposed individuals exhibit variable susceptibility to benzene effect that arises, in part, from genetic variations in benzene metabolism, DNA repair, genomic stability, and immune function. These new studies of benzene leukemogenesis and hematotoxicity are expected to provide insights into how environmental and occupational chemicals affect stem cells to cause cancer and toxicity, which impact the risk assessment, permissible level, and therapy of benzene exposure.     

Toxic effects of benzene and benzene metabolites on granulopoietic stem cells and bone marrow cellularity in mice

Mice were injected subcutaneously with benzene or one of three of its metabolites (phenol, hydroquinone, or 1,2-dihydro-1,2-dihydroxybenzene). The adverse effects on the concentration of granulopoietic stem cells (measured as number of colony-forming units per tibia or per 10⁵ cells) and on the bone marrow cellularity in tibia were measured. Benzene had strong toxic effects. Thus, 0.7 mg benzene/kg body wt injected daily on 6 consecutive days gave detectable effects on stem cell concentration, and 3.5 mg/kg/day affected also cellularity. Six daily injections of 440 mg benzene/kg reduced cellularity and number of colony-forming units per tibia by 86–95%. None of the benzene metabolites tested could reproduce the strong effects of benzene when injected subcutaneously, although phenol slightly but significantly affected stem cell concentration. Toluene, a competitive inhibitor of benzene metabolism, significantly alleviated the effects of benzene. Regeneration of the bone marrow after benzene injections occurred rapidly during the first week, then at a slower rate for the next 4 weeks. At this time cellularity and granulopoietic stem cell concentration were restored, but the fraction of stem cells in S phase was still higher than in controls, indicating a still elevated proliferation rate.     

Subclinical effects of groundwater contaminants. IV. Effects of repeated oral exposure to combinations of benzene and toluene on regional brain monoamine metabolism in mice

Benzene and toluene are known neurotoxicants that may interact in vivo. The effect of combined treatment with benzene and toluene on the endogenous concentrations of the catecholamines norepinephrine (NE) and dopamine (DA), the catecholamine metabolites vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the indoleamine serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), were investigated in six discrete brain regions of CD-1 mice. Groups of male, adult mice were continuously exposed to benzene (166 mg/l), toluene (80 and 325 mg/l), and combinations of benzene + toluene (80 or 325 mg/l) in drinking water for 4 weeks. Benzene produced increases of NE in the hypothalamus, cortex, midbrain and medulla oblongata, DA in the hypothalamus and corpus striatum, and 5-HT in all dissected brain regions except cerebellum. Elevated levels of various monoamine metabolites were also observed in these brain areas. Toluene ingestion alone also significantly increased the concentrations of NE, DA, 5-HT, and their metabolites in several brain regions. Mice given the combined treatments exhibited raised regional neurochemical levels when compared to the untreated controls. Increased concentrations of biogenic amine metabolites in several brain regions were greater in the combined exposures of benzene and toluene than when either chemical was used alone. The findings were different from those observed on immune parameters using similar treatment protocols, where simultaneous exposure to toluene prevented the immunotoxic effects of benzene.     

Tracking stem cells for cellular therapy in stroke

Stem cell transplantation has emerged as a promising treatment strategy for stroke. The development of effective ways to monitor transplanted stem cells is essential to understand how stem cell transplantation enhances stroke recovery and ultimately will be an indispensable tool for advancing stem cell therapy to the clinic. In this review, we describe existing methods of tracking transplanted stem cells in vivo, including optical imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET), with emphasis on the benefits and drawbacks of each imaging approach. Key considerations such as the potential impact of each tracking system on stem cell function, as well as its relative applicability to humans are discussed. Finally, we describe multi-modal imaging strategies as a more comprehensive method to track transplanted stem cells in the stroke-injured brain.     

Adult neural stem cell therapy: expansion in vitro, tracking in vivo and clinical transplantation

Neural stem cells (NSCs) are present not only in the developing nervous systems, but also in the adult human central nervous system (CNS). It is long thought that the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus are the main sources of human adult NSCs, which are considered to be a reservoir of new neural cells. Recently adult NSCs with potential neural capacity have been isolated from white matter and inferior prefrontal subcortex in the human brain. Rapid advances in the stem cell biology have raised appealing possibilities of replacing damaged or lost neural cells by transplantation of in vitro-expanded stem cells and/or their neuronal progeny. However, sources of stem cells, large scale expansion, control of the differentiations, and tracking in vivo represent formidable challenges. In this paper we review the characteristics of the adult human NSCs, their potentiality in terms of proliferation and differentiation capabilities, as well as their large scale expansion for clinical needs. This review focuses on the major advances in brain stem cell-based therapy from the clinical perspective, and summarizes our work in clinical phase I-II trials with autologuous transplantation of adult NSCs for patients with open brain trauma. It also describes multiple approaches to monitor adult human NSCs labeled superparamagnetic nanoparticles after transplantation and explores the intriguing possibility of stem cell transplantation.     

High MR sensitive fluorescent magnetite nanocluster for stem cell tracking in ischemic mouse brain

Stem cells have shown a great potential to treat diseases and injuries, including ischemic brain injury. However, developing agents for the long-term tracking of stem cells with few side effects is still challenging. Our aim is to develop a novel fluorescent-magnetite-nanocluster (FMNC) with high MRI sensitivity and to examine its application in the labeling and tracking of mesenchymal stem cells (MSC). For this purpose, we developed FMNC by embedding individual magnetite nanoparticles (NPs) into a polystyrene scaffold coated with two layers of silica and a sandwiched layer of rhodamine. We examined the efficacy of FMNC in MSC labeling and the feasibility of tracking FMNC-labeled MSCs in the ischemic mouse brain. We found that FMNC has high cell-labeling efficiency with no adverse effects on MSCs. In a mouse middle cerebral artery occlusion model, FMNC-labeled MSCs migrated to and accumulated in the ischemic region after FMNC-labeled MSC transplantation. MRI findings highly correlated to immunohistochemistry results. FROM THE CLINICAL EDITOR: In this study, the authors report a novel fluorescent-magnetite-nanocluster with high MRI sensitivity and to labeling and tracking of mesenchymal stem cells, and provide in vivo data utilizing a murine stroke model.     

The importance of pluripotential stem cells in benzene toxicity

Several schedules of benzene exposure were evaluated for their effects on peripheral white blood cell counts, bone marrow cellularity and transplantable colony forming units (CFU-S) in male C57 B1/6 mice. Intermittent exposure to 4000 ppm benzene in air produced leukopenia without altering the bone marrow cellularity. This same treatment, however, decreased the number of CFU-S to 30% of control values. Uninterrupted exposure to lower levels of benzene decreased peripheral cell counts within24 h, and later decreased marrow cellularity. Exposure of a non-dividing population of stem cells (CFU-S) to benzene for up to 24 h produced no detectable effect on the subsequent development of spleen colonies, suggesting that the effect of benzene on CFU-S occurs only after peripheral cells are depleted. These findings indicate that benzene has affects on both differentiated cells and undifferentiated stem cells. An effect on the pluripotential stem cell is an important aspect of benzene toxicity, but not its exclusive or initial site of action.     

Oxidative phosphorylation in different regions of the rat brain following morphine administration]

The influence exercised by morphine in a dose of 20 mg/kg, introduced intraperitoneally, and also in concentrations of 10(-3) and 10(-5) "in vitro" on the parameters of oxidative phosphorylation of the brain cortex and stem of rats was studied. Morphine, used in a concentration of 10(-3), is shown to speed up the substrates oxidation rate. During the first days of its administration the narcotic analgetic inhibited oxidation of mitochondia released from the brain stem, and, once habituation to the narcotic had developed, the inhibition ceased to be effective. In "in vivo" experiments and in vitro tests the effect of phosphorylation remained unchanged. The data obtained suggest that with developing habituation in regard to morphine the functions of the brain stem and cortex mitochondria do not undergo any substantial changes.     

Effect of phenobarbital pretreatment on benzene biotransformation in the rat

Factors responsible for different quantitative effect of phenobarbital (PB) pretreatment (sodium phenobarbital, 50 mg kg^–1 day^–1 for 3 days) on benzene metabolism to phenol in vivo and in vitro were studied in male Wistar rats. A more than 4-fold increase of benzene metabolism was observed with 9,000 g supernatant of liver homogenate, 2.8- to 4-fold increase with isolated perfused liver; phenol formation in vivo after oral benzene was increased by PB 2-fold, but only shortly following benzene administration and the enhancement rapidly diminished to 1.15-fold increase in the total excreted phenol. Benzene concentrations in 9,000 g supernatant incubations were 2 mM, those with isolated perfused livers were up to 4 mM, but those in blood in vivo were below 0.3 mM; the effect of PB induction in vivo disappeared along with decreasing benzene and increasing phenol blood concentrations which surpassed benzene 2–3 h after oral benzene administration. The effect of benzene concentration on the manifestation of PB induction is also supported by almost a 2-fold increased phenol formation in PB rats over controls in vivo after repeated administration of benzene. The elimination of radioactive metabolites of orally administered benzene-¹⁴C, 3 mmoles kg^–1, in urine was markedly inhibited by intraperitoneal administration of phenol (1.2 mmole kg^–1), but not by pyrocatechol, resorcinol or hydroquinol (0.6 mmole kg^–1, respectively) suggesting that phenol might inhibit benzene metabolism in vivo especially when its concentration exceeds that of benzene.     

Effects of 1,1,1-trichloroethane on the cGMP metabolism in mouse brain

Administration of 1,1,1-trichloroethane (TCE) to mice by inhalation or intraperitoneally reduced the cGMP contents of the brain stem, cerebral cortex, and vermis anterior, including the hemispheres. Following intraperitoneal administration the cGMP contents of the vermis posterior and hippocampus were also reduced. To investigate the mechanism underlying these changes, the effects of TCE on brain guanylate cyclase (GC) and phosphodiesterase (PDE) activities were examined after intraperitoneal administration in mice. The basal GC activities in the particulate and soluble fractions of the homogenates of the cerebellum, brain stem and cerebral cortex were not altered by TCE. In the cerebellum TCE treatment inhibited sodium azide-stimulated GC activity in the particulate and soluble fractions, while in the brain stem it enhanced the particulate GC activity induced by Ca2+. TCE treatment increased the rate of cGMP hydrolysis in the cerebral cortex and this was further accelerated by addition of Ca2+. Ca2+ also increased the rate of cGMP hydrolysis in the brain stem. However, in the cerebellum TCE enhanced the Ca2+-independent PDE activity as well as the enzyme activity in the presence of Ca2+ and exogenous calmodulin. These results indicate that the reduction of the cGMP content in the brain stem and cerebral cortex in vivo on exposure to TCE is due to changes in the rate of cGMP hydrolysis. In the cerebellum the TCE reduced cGMP content may be regulated by increased rate of cGMP hydrolysis as well as effects on the guanylate cyclase.     

苯丁酸钠对SHG-44 胶质细胞瘤株的抗肿瘤作用机制研究

目的观察苯丁酸钠对人脑胶质细胞瘤株SHG-44的抗肿瘤作用,探讨其抗肿瘤作用的机制是否涉及调节ASIC2的表达。方法体外培养人脑胶质细胞瘤株SHG-44,平皿克隆形成法测定细胞描定依赖性生长能力,PI染色流式细胞术（Flow cytometry,FCM）分析细胞周期和凋亡率,Hoechst33258染色荧光显微镜观察凋亡细胞的形态,Western Bloting分析药物对ASIC2蛋白表达的影响。结果平皿集落形成法检测显示苯丁酸钠对人脑胶质细胞瘤株SHG-44的抗肿瘤作用,且呈浓度依赖性。PI染色FCM结果表明苯丁酸钠以时间和浓度依赖方式对人脑胶质细胞瘤株SHG-44的抗肿瘤作用。Hoechst33258染色荧光显微镜观察可见核染色质凝集,凋亡细胞呈致密浓染,与对照组相比,苯丁酸钠处理后凋亡细胞比例增加,且呈时间和浓度依赖性。Western Bloting检测显示苯丁酸钠诱导细胞凋亡的机制涉及上调ASIC2表达。结论苯丁酸钠在体外对人脑胶质细胞瘤株SHG-44具有抑制增值的作用,初步推断苯丁酸钠诱发人脑胶质细胞凋亡与其上调ASIC2表达有关。     

Effects of lead,cadmium and mercury on brain adenylate cyclase

The effects of lead, cadmium and mercury ions on adenylate cyclase activity of rat cerebrum, cerebellum and brain stem were studied in vitro and in vivo. Adenylate cyclase activity in homogenates of cerebellum as well as cerebrum and brain stem was found to be inhibited by micromolar concentrations of these heavy metal ions in vitro. Administration of lead acetate trihydrate (25 mg/kg body wt i.v.) produced an initial increase of adenylate cyclase activity in the cerebellum and brain stem 1 h after injection, followed by a significant decrease of enzyme activity in cerebrum and cerebellum 4 h after the injection. Chronic lead treatment achieved by feeding lead containing diets, which generated blood lead levels of 31.3 ± 3.8, 68.8 ± 1.5 and 121.5 ± 8.6 μg Pb/100 g blood resp., produced a significant increase of brain lead levels and a 10–30% reduction of adenylate cyclase activity in cerebrum, cerebellum and brain stem. Phosphodiesterase activity was reduced under these conditions in the range of 10–20% in cerebellum and brain stem, but not in cerebrum.     

Effect of phenobarbital pretreatment on in vitro enzyme kinetics and in vivo biotransformation of benzene in the rat

Phenobarbital pretreatment (50 mg/kg/day for 3 days orally) of male Wistar rats increased V _max of benzene in vitro hepatic microsomal biotransformation about 6-fold without changing K _m . However, benzene blood levels after oral, intraperitoneal, or subcutaneous benzene administration (3–3.5 mmoles/kg) were not influenced by phenobarbital pretreatment. The phenol blood levels after oral or intraperitoneal benzene were increased by phenobarbital pretreatment, but less than expected from in vitro data and only 3 h after benzene administration. Phenol elimination in urine after subcutaneous benzene was not affected by phenobarbital. After oral or intraperitoneal benzene administration, phenol urine excretion closely followed the levels of phenol in blood, i.e., rate of phenol urine excretion was significantly, but shortly increased, and the cumulative urine excretion of phenol increased very little or remained unchanged. Differences between the in vitro and in vivo observations of the effect of phenolbarbital on benzene biotransformation may partly be explained by distribution of benzene, which apparently limited benzene availability for biotransformation (V _d =5.5) and caused rapid decrease of benzene concentrations in blood. Conditions for enzyme activity may have been substantially different in vitro vs. in vivo: in vitro concentrations of benzene were at least by an order of magnitude higher than phenol concentrations, while in vivo, an opposite relation prevailed making a competition for microsomal monooxygenase possible. Cofactor availability may be another rate-limiting step or factor of in vivo benzene biotransformation, as benzene ring hydroxylation requires high energy. The rate of in vitro hepatic microsomal benzene biotransformation proved to be of limited value when predicting benzene quantitative biotransformation in vivo in contradistinction to various substrates where the in vitro and in vivo biotransformation data are in good agreement     

Endogenous and exogenous CNS derived stem/progenitor cell approaches for neurotrauma

Neural stem/progenitor cells capable of generating new neurons and glia, reside in specific areas of the adult mammalian central nervous system (CNS), including the ependymal region of the spinal cord and the subventricular zone (SVZ), hippocampus, and dentate gyrus of the brain. Much is known about the neurogenic regions in the CNS, and their response to various stimuli including injury, neurotrophins (NFs), morphogens, and environmental factors like learning, stress, and aging. This work has shaped our current views about the CNS's potential to recover lost tissue and function post-traumatically and the therapies to support the intrinsic regenerative capacity of the brain or spinal cord. Recently, intensive research has explored the potential of harvesting, culturing, and transplanting neural stem/progenitors as a therapeutic intervention for spinal cord injury (SCI) and traumatic brain injury (TBI). Another strategy has focused on maximizing the potential of this endogenous population of cells by stimulating their recruitment, proliferation, migration, and differentiation in vivo following traumatic lesions to the CNS. The promise of such experimental treatments has prompted tissue and biomaterial engineers to implant synthetic three-dimensional biodegradable scaffolds seeded with neural stem/progenitors into CNS lesions. Although there is no definitive answer about the ideal cell type for transplantation, strong evidence supports the use of region specific neural stem/progenitors. The technical and logistic considerations for transplanting neural stem/progenitors are extensive and crucial to optimizing and maintaining cell survival both before and after transplantation, as well as for tracking the fate of transplanted cells. These issues have been systematically addressed in many animal models, that has improved our understanding and approach to clinical therapeutic paradigms.     

Dermal absorption of benzene in occupational settings: Estimating flux and applications for risk assessment

There is growing emphasis in the United States and Europe regarding the quantification of dermal exposures to chemical mixtures and other substances. In this paper, we determine the dermal flux of benzene in neat form, in organic solvents, and in aqueous solutions based on a critical review and analysis of the published literature, and discuss appropriate applications for using benzene dermal absorption data in occupational risk assessment. As part of this effort, we synthesize and analyze data for 77 experimental results taken from 16 studies of benzene skin absorption. We also assess the chemical activity of benzene in simple hydrocarbon solvent mixtures using a thermodynamic modeling software tool. Based on the collective human in vivo, human in vitro, and animal in vitro data sets, we find that the steady-state dermal flux for neat benzene (and benzene-saturated aqueous solutions) ranges from 0.2 to 0.4?mg/(cm²?h). Observed outlier values for some of the animal in vivo data sets are possibly due to the use of test species that have more permeable skin than humans or study conditions that resulted in damage to the skin barrier. Because relatively few dermal absorption studies have been conducted on benzene-containing organic solvents, and available test results may be influenced by study design or vehicle effects, it is not possible to use these data to quantify the dermal flux of benzene for other types of solvent mixtures. However, depending on the application, we describe several potential approaches that can be used to derive a rough approximation of the steady-state benzene dermal flux for these mixtures. Important limitations with respect to quantifying and evaluating the significance of dermal exposures to benzene in occupational settings include a lack of data on (1) factors that affect the dermal uptake of benzene, (2) the dermal flux of benzene for different organic solvent mixtures, (3) meaningful metrics for evaluating the dermal uptake of benzene, (4) steady-state versus non-steady-state dermal flux values for benzene, (5) the effect of skin damage on the dermal flux of benzene, (6) standardized test methods for estimating the dermal flux of benzene, and (7) robust estimates of the evaporation rate of benzene from different liquid vehicles.     

Xenobiotic metabolism and the mechanism(s) of benzene toxicity

The investigation of the mechanism(s) of benzene toxicity/leukemogenesis over the past 50 years has been contemporaneous with developments in the study of xenobiotic metabolism. Research on the cytochrome P450 (CYP) enzyme system, and related systems in vivo and in vitro, which culminated in the isolation and reconstitution of the many CYPs, established pathways for the study of xenobiotic metabolism and its relationship to the biological activity of many chemicals. The essential role for metabolism of benzene as a precursor to the demonstration of benzene toxicity led to extensive studies of benzene metabolism, many of which will be reviewed here. Benzene toxicity/leukemogenesis, however, is a function of the bone marrow, a site remote from the liver where most benzene metabolism occurs. Studies of benzene metabolism have delineated the array of metabolites which appear to play a role in bone marrow damage, but further studies, both in vivo and in vitro, using appropriate animal models, will be needed to fully understand the impact of benzene and its metabolites on bone marrow function.     

A review of quantitative studies of benzene metabolism

Benzene is a ubiquitous, highly flammable, colorless liquid that is a known hematotoxin, myelotoxin, and human leukemogen. Benzene-induced toxicity in animals is clearly mediated by its metabolism. The mechanisms of acute hemato- and myelotoxicity in humans are almost certainly the same as in animals, and there is compelling evidence that metabolism is requisite for the induction of leukemia in humans. A very large number of experimental investigations of benzene metabolism have been conducted with animals, both in vivo and in vitro. There have also been many investigations of benzene metabolism in humans and with human tissues, Although the blood or tissue concentrations of benzene metabolites in humans resulting from benzene exposure have never been measured. Further, a number of mathematical models of benzene metabolism and dosimetry have been developed. In this article, we consider results from both experimental and mathematical modeling research, with particular emphasis on the last decade, and discuss the factors that are likely to be most influential in the metabolism of benzene.     

Identification of N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol, and hydroquinone

The metabolite 2-(S-glutathionyl)hydroquinone is formed when a microsomal incubation mixture containing either benzene or phenol is supplemented with glutathione. This metabolite is derived from the conjugation of benzoquinone, an oxidation product of hydroquinone. However, neither the glutathione conjugate or its mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, have been identified as metabolites resulting from in vivo metabolism of benzene, phenol, or hydroquinone. To determine if a hydroxylated mercapturate is produced in vivo, we treated male Sprague-Dawley rats with either benzene (600 mg/kg), phenol (75 mg/kg), or hydroquinone (75 mg/kg) and collected the urine for 24 hr. HPLC coupled with electrochemical detection confirmed the presence of a metabolite that was chromatographically and electrochemically identical to N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine. The metabolite was isolated from the urine samples and treated with diazomethane to form the N-acetyl-S-(2,5-dimethoxyphenyl)-L-cysteine methyl ester derivative. The mass spectra obtained from these samples were identical to that of an authentic sample of the derivative. The results of these experiments indicate that benzene, phenol, and hydroquinone are metabolized in vivo to benzoquinone and excreted as the mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine.     

Dermal absorption of benzene in occupational settings: estimating flux and applications for risk assessment

There is growing emphasis in the United States and Europe regarding the quantification of dermal exposures to chemical mixtures and other substances. In this paper, we determine the dermal flux of benzene in neat form, in organic solvents, and in aqueous solutions based on a critical review and analysis of the published literature, and discuss appropriate applications for using benzene dermal absorption data in occupational risk assessment. As part of this effort, we synthesize and analyze data for 77 experimental results taken from 16 studies of benzene skin absorption. We also assess the chemical activity of benzene in simple hydrocarbon solvent mixtures using a thermodynamic modeling software tool. Based on the collective human in vivo, human in vitro, and animal in vitro data sets, we find that the steady-state dermal flux for neat benzene (and benzene-saturated aqueous solutions) ranges from 0.2 to 0.4 mg/(cm2·h). Observed outlier values for some of the animal in vivo data sets are possibly due to the use of test species that have more permeable skin than humans or study conditions that resulted in damage to the skin barrier. Because relatively few dermal absorption studies have been conducted on benzene-containing organic solvents, and available test results may be influenced by study design or vehicle effects, it is not possible to use these data to quantify the dermal flux of benzene for other types of solvent mixtures. However, depending on the application, we describe several potential approaches that can be used to derive a rough approximation of the steady-state benzene dermal flux for these mixtures. Important limitations with respect to quantifying and evaluating the significance of dermal exposures to benzene in occupational settings include a lack of data on (1) factors that affect the dermal uptake of benzene, (2) the dermal flux of benzene for different organic solvent mixtures, (3) meaningful metrics for evaluating the dermal uptake of benzene, (4) steady-state versus non-steady-state dermal flux values for benzene, (5) the effect of skin damage on the dermal flux of benzene, (6) standardized test methods for estimating the dermal flux of benzene, and (7) robust estimates of the evaporation rate of benzene from different liquid vehicles.     

Adult neurogenesis: can analysis of cell cycle proteins move us "Beyond BrdU"?

One of the greatest scientific discoveries of the 20th century is that the mammalian brain can give rise to new neurons throughout the lifespan. The phenomenon of adult neurogenesis raises hopes of harnessing neural stem cell for brain repair, and has sparked interest in novel roles for these new neurons, such as olfaction, spatial memory, and even regulation of mood. Traditionally, studies on adult neurogenesis have relied on exogenous markers of DNA synthesis, such as bromodeoxyuridine (BrdU), to label and track the birth of new cells. However, the exponential increase in our knowledge of endogenous markers of cycling cells has ushered in a new era of stem cell biology. Here we review the strides made in using endogenous cell cycle proteins to study adult neurogenesis in vivo. We (1) discuss the distribution of endogenous cell cycle proteins in proliferative regions of the adult mammalian brain; (2) review cell cycle phase-specific information gained from analyzing a combination of endogenous cell cycle proteins; and (3) provide data on the regulation of cell cycle proteins by a robust inhibitor of proliferation, morphine. The ability of BrdU to birthdate cells ensures it will always serve a role in studies of adult neurogenesis, thus preventing us from moving entirely 'beyond BrdU'. However, it is hoped that this review will provide interested researchers with the tools needed to apply the powerful and relatively novel approach of analyzing endogenous cell cycle proteins to the study of stem cells in general and adult neurogenesis in particular.