Efficient Design to Monitor the Site-specific Sustained Release of a Non-Emissive Anticancer Drug.

A pH-responsive smart nanocarrier with significant components was synthesized by conjugating the non-emissive anticancer drug methyl orange and polyethylene glycol derived folate moiety to the backbone of polynorbornene. Complete synthesis procedure and characterization methods of three monomers included in the work: norbornene-derived Chlorambucil (Monomer 1), norbornene grafted with polyethylene glycol, and folic acid (Monomer 2) and norbornene attached methyl orange (Monomer 3) connected to the norbornene backbone through ester linkage were clearly discussed. Finally, the random copolymer CHO PEG FOL METH was synthesized by ring-opening metathesis polymerization (ROMP) using Grubbs' second-generation catalyst. Advanced polymer chromatography (APC) was used to find the final polymer's molecular weight and polydispersity index (PDI). Dynamic light scattering, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were utilized to explore the prodrug's size and morphology. Release experiments of the anticancer drug, Chlorambucil and the coloring agent, methyl orange, were performed at different pH and time. Cell viability assay was carried out for determining the rate of survived cells, followed by the treatment of our final polymer named CHO PEG FOL METH.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines.

Vesicants cause a multitude of cutaneous reactions like erythema, blisters and ulcerations. After exposure to sulfur mustard (SM) and related compounds, patients present dermal symptoms typically known for chemicals categorized as skin sensitizer (e.g. hypersensitivity and flare-up phenomena). However, although some case reports led to the assumption that SM and other alkylating compounds represent sensitizers, a comprehensive investigation of SM-triggered immunological responses has not been conducted so far. Based on a well-structured system of in chemico and in vitro test methods, the Organization for Economic Co-operation and Development (OECD) established procedures to categorize agents on their skin sensitizing abilities. In this study, the skin sensitizing potential of SM and three related alkylating agents (AAs) was assessed following the OECD test guidelines. Besides SM, investigated AAs were chlorambucil (CHL), nitrogen mustard (HN3) and 2-chloroethyl ethyl sulfide (CEES). The methods are described in detail in the EURL ECVAM DataBase service on ALternative Methods to animal experimentation (DB-ALM). In accordance to OECD recommendations, skin sensitization is a pathophysiological process starting with a molecular initiating step and ending with the in vivo outcome of an allergic contact dermatitis. This concept is called adverse outcome pathway (AOP). An AOP links an adverse outcome to various key events which can be assayed by established in chemico and in vitro test methods. Positive outcome in two out of three key events indicates that the chemical can be categorized as a skin sensitizer. In this study, key event 1 "haptenation" (covalent modification of epidermal proteins), key event 2 "activation of epidermal keratinocytes" and key event 3 "activation of dendritic cells" were investigated. Covalent modification of epidermal proteins measured by using the DPRA-assay provided distinct positive results for all tested substances. Same outcome was seen in the KeratinoSens assay, investigating the activation of epidermal keratinocytes. The h-CLAT assay performed to determine the activation of dendritic cells provided positive results for SM and CEES but not for CHL and HN3. Altogether, following OECD requirements, our results suggest the classification of all investigated substances as skin sensitizers. Finally, a tentative AOP for SM-induced skin sensitization is suggested.

Construction of drug-drug conjugate supramolecular nanocarriers based on water-soluble pillar[6]arene for combination chemotherapy.

The synergistic effect of two anticancer drugs can significantly overcome the multidrug resistance of tumor cells and improve the drug bioavailability. Herein, two different anticancer drugs, camptothecin and chlorambucil, are successfully connected together by a disulfide linkage to get a novel drug-drug conjugated prodrug (G). Using water-soluble pillar[6]arene (WP6) as a host molecule, a supramolecular host-guest complex WP6⊃G is formed, which can further self-assemble into supramolecular vesicles in aqueous solution. In the specific microenvironment of cancer cells, the disulfide linkage is destroyed and the two anticancer drugs can be released efficiently to achieve a better synergistic effect than a single anticancer drug. Notably, these prodrug nanocarriers can not only effectively kill the cancer cells but also obviously reduce the undesirable side effects on normal cells.

A statistical approach for analyzing data from the in vivo Pig-a gene mutation assay.

The in vivo Pig-a gene mutation assay serves to evaluate the genotoxic potential of chemicals. In the rat blood-based assay, the lack of CD59 on the surface of erythrocytes is quantified via fluorophore-labeled antibodies in conjunction with flow cytometric analysis to determine the frequency of Pig-a mutant phenotype cells. The assay has achieved regulatory relevance as it is suggested as an in vivo follow-up test for Ames mutagens in the recent ICH M7 [25] step 4 document. However, very little work exists regarding suitable statistical approaches for analyzing Pig-a data. In the current report, we present a statistical strategy based on a two factor model involving 'treatment' and 'time' incl. their interaction and a baseline covariate for log proportions to compare treatment and vehicle data per time point as well as in time. In doing so, multiple contrast tests allow us to discover time-related changes within and between treatment groups in addition to multiple treatment comparisons to a control group per single time point. We compare our proposed strategy with the results of classical Dunnett and Wilcoxon-Mann-Whitney tests using two data sets describing the mode of action of Chlorambucil and Glycidyl methacrylate both analyzed in a 28-day treatment schedule.

A Multi-Mitochondrial Anticancer Agent that Selectively Kills Cancer Cells and Overcomes Drug Resistance.

Mitochondria are double-membrane-bound organelles involved mainly in supplying cellular energy, but also play roles in signaling, cell differentiation, and cell death. Mitochondria are implicated in carcinogenesis, and therefore dozens of lethal signal transduction pathways converge on these organelles. Accordingly, mitochondria provide an alternative target for cancer management. In this study, F16, a drug that targets mitochondria, and chlorambucil (CBL), which is indicated for the treatment of selected human neoplastic diseases, were covalently linked, resulting in the synthesis of a multi-mitochondrial anticancer agent, FCBL. FCBL can associate with human serum albumin (HSA) to form an HSA-FCBL nanodrug, which selectively recognizes cancer cells, but not normal cells. Systematic investigations show that FCBL partially accumulates in cancer cell mitochondria to depolarize mitochondrial membrane potential (MMP), increase reactive oxygen species (ROS), and attack mitochondrial DNA (mtDNA). With this synergistic effect on multiple mitochondrial components, the nanodrug can effectively kill cancer cells and overcome multiple drug resistance. Furthermore, based on its therapeutic window, HSA-FCBL exhibits clinically significant differential cytotoxicity between normal and malignant cells. Finally, while drug dosage and drug resistance typically limit first-line mono-chemotherapy, HSA-FCBL, with its ability to compromise mitochondrial membrane integrity and damage mtDNA, is expected to overcome those limitations to become an ideal candidate for the treatment of neoplastic disease.

Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay.

Three-Arm, Biotin-Tagged Carbazole-Dicyanovinyl-Chlorambucil Conjugate: Simultaneous Tumor Targeting, Sensing, and Photoresponsive Anticancer Drug Delivery.

The design, synthesis, and in vitro biological studies of a biotin-carbazole-dicyanovinyl-chlorambucil conjugate (Bio-CBZ-DCV-CBL; 6) are reported. This conjugate (6) is a multifunctional single-molecule appliance composed of a thiol-sensor DCV functionality, a CBZ-derived phototrigger as well as fluorescent reporter, and CBL as the anticancer drug, and Bio as the cancer-targeting ligand. In conjugate 6, the DCV bond undergoes a thiol-ene click reaction at pH<7 with intracellular thiols, thereby shutting down internal charge transfer between the donor CBZ and acceptor DCV units, resulting in a change of the fluorescence color from green to blue, and thereby, sensing the tumor microenvironment. Subsequent photoirradiation results in release of the anticancer drug CBL in a controlled manner.

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels - either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention. We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Modified in vivo comet assay detects the genotoxic potential of 14-hydroxycodeinone, an α,ß-unsaturated ketone in oxycodone.

14-Hydroxycodeinone (14-HC) is an α,ß-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay.

Ring-opening polymerization of prodrugs: a versatile approach to prepare well-defined drug-loaded nanoparticles.

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release the drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s)/initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a poly(ethylene glycol) macroinitiator results in amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 µM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 µM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 µM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

Scopine as a novel brain-targeting moiety enhances the brain uptake of chlorambucil.

The blood brain barrier (BBB) represents the biggest challenge for therapeutic drugs to enter the brain. In our study, we selected chlorambucil (CHL), an alkylating agent, as the model therapeutic agent, and used scopine as a novel brain-targeting moiety. Here, we synthesized Chlorambucil-Scopine (CHLS) prodrug and evaluated its brain-targeting efficacy. The tissue distribution study after i.v. injection revealed that the AUC0-t and Cmax of CHLS in the brain were 14.25- and 12.20-fold of CHL, respectively. Specifically, CHLS accumulated in bEnd.3 and C6 cells in an energy-dependent manner. In C6 cells, superior anti-glioma activity with a significantly decreased IC50 of 65.42 nM/mL was observed for CHLS compared to CHL (IC50 > 400 nM/mL). The safety evaluation, including acute toxicity, pathology, and hematology study, showed minimal toxicity toward nontargeting tissues, and also reached a lower systemic toxicity at 5 mg/kg (i.v.). Our results suggested that scopine is a potential brain-targeting moiety for enhancing the brain uptake efficiency of CHL.

Chlorambucil (nitrogen mustard) induced impairment of early vascular endothelial cell migration - effects of α-linolenic acid and N-acetylcysteine.

Alkylating agents (e.g. sulfur and nitrogen mustards) cause a variety of cell and tissue damage including wound healing disorder. Migration of endothelial cells is of utmost importance for effective wound healing. In this study we investigated the effects of chlorambucil (a nitrogen mustard) on early endothelial cells (EEC) with special focus on cell migration. Chlorambucil significantly inhibited migration of EEC in Boyden chamber and wound healing experiments. Cell migration is linked to cytoskeletal organization. We therefore investigated the distribution pattern of the Golgi apparatus as a marker of cell polarity. Cells are polarized under control conditions, whereas chlorambucil caused an encircling perinuclear position of the Golgi apparatus, indicating non-polarized cells. ROS are discussed to be involved in the pathophysiology of alkylating substances and are linked to cell migration and cell polarity. Therefore we investigated the influence of ROS-scavengers (α-linolenic acid (ALA) and N-acetylcysteine (NAC)) on the impaired EEC migration. Both substances, in particular ALA, improved EEC migration. Notably ALA restored cell polarity. Remarkably, investigations of ROS and RNS biomarkers (8-isoprostane and nitrotyrosine) did not reveal a significant increase after chlorambucil exposure when assessed 24h post exposure. A distinct breakdown of mitochondrial membrane potential (measured by TMRM) that recovered under ALA treatment was observed. In conclusion our results provide compelling evidence that the alkylating agent chlorambucil dramatically impairs directed cellular migration, which is accompanied by perturbations of cell polarity and mitochondrial membrane potential. ALA treatment was able to reconstitute cell polarity and to stabilize mitochondrial potential resulting in improved cell migration.

Photoresponsive quinoline tethered fluorescent carbon dots for regulated anticancer drug delivery.

A photoresponsive nano drug delivery system (DDS) was constructed using two new ingredients: fluorescent carbon dots and a quinoline based phototrigger. The strong fluorescent properties of carbon dots have been explored for in vitro cellular imaging application, and the phototrigger ability of quinoline was exploited for efficient anticancer drug release using both one-photon and two-photon excitation.

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins.

Previous findings showed that the anticancer drugs p-N,N-bis(2-chloroethyl) amino-l-phenylalanine (melphalan, MEL) and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin-V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of ß- and γ-tubulin in the presence of pan-caspase inhibitor. The expressions of Aurora-A, Aurora-B, survivin and γ-tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora-A, Aurora-B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil.

Design, synthesis and biological evaluation of estradiol-chlorambucil hybrids as anticancer agents.

A series of estradiol-chlorambucil hybrids was synthesized as anticancer drugs for site-directed chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of estrone at position 16alpha of the steroid nucleus. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent breast cancer cell lines. The novel hybrids showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals the influence of the length of the spacer chain between carrier and drug molecule.

Time-course study of the immunotoxic effects of the anticancer drug chlorambucil in the rat.

In 2005, the International conference on harmonization (ICH) recommended that all new human pharmaceuticals be tested for unintended immunomodulatory potential via a tiered approach. Included in this approach is a semiquantitative description of changes in the separate compartments of lymphoid tissue (also called enhanced histopathology). Chlorambucil was administered to Hanover Wistar rats at regular time points, followed by a treatment-free (recovery) period. Groups of treated and control animals were sacrificed regularly during both the treatment and recovery periods. Selected tissues were removed, weighed fresh and fixed in formalin, processed, and stained with hematoxylin and eosin. Blood samples and bone marrow smears were also obtained. With the use of enhanced histopathology, a description of the changes in lymphoid tissues and bone marrow was used as a means of assessing the susceptibility, and recovery, of the different lymphoid cell populations over time. A correlation with organ weights, flow cytometry data, and bone marrow cytology was achieved. The administration of chlorambucil in the Hanover Wistar rat provided a useful tool to examine the rate and sequence of changes in the lymphoid organs and bone marrow during treatment with, and the recovery from the effects of, a potent immunosuppressive agent.